Chimeric hepatocyte growth factor (HGF) ligand variants

ABSTRACT

The invention concerns a method for activating receptors selected from receptor tyrosine kinases, cytokine receptors and members of the nerve growth factor receptor superfamily. A conjugate comprising the direct fusion of at least two ligands capable of binding to the receptor(s) to be activated is contacted with the receptors, whereby the ligands bind their respective receptors inducing receptor oligomerization.

This application is a continuation of application Ser. No. 08/087,784filed as PCT/US93/04717 May 17, 1993, now abandoned, which is a Section371 application of PCT/US93/04717 filed on 17 May 1993, which is acontinuation-in-part application of Ser. No. 07/950,572 filed on 21 Sep.1992, now abandoned, which is a continuation-in-part application of Ser.No. 07/884,811 filed on 18 May 1992 (issued as U.S. Pat. No. 5,316,921)and Serial No. 07/885,971 filed on 18 May 1992 (issued as U.S. Pat. No.5,328,837), which applications are incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to a method for receptor activation. Moreparticularly, the invention concerns a method for ligand-inducedoligomerization of cell-surface receptors. The invention further relatesto methods for making ligand variants that act as competitive agonistsof the respective native ligands, and to ligand-immunoglobulin chimeras.

BACKGROUND OF THE INVENTION

Many polypeptides, such as growth factors, differentiation factors, andhormones mediate their actions by binding to and activating cell surfacereceptors. Although the mechanism of receptor activation varies forspecific receptor-ligand pairs and is often not entirely understood, itis a common feature of many receptors that they need to be oligomerizedto become active, or that their activity is enhanced by oligomerization.Growth factor receptors with tyrosine kinase activity (receptor tyrosinekinases) and certain cytokine receptors are typical representatives ofsuch receptors.

Receptors with tyrosine kinase activity have a similar moleculartopology. They all possess an extracellular ligand binding domain, ahydrophobic transmembrane domain, and a cytoplasmic domain that containsa tyrosine kinase catalytic domain, and can be further classified on thebasis of sequence similarity and distinct structural characteristics(Hanks, S. K. et al., Science 241, 42-52 (1988); Yarden, Y. and Ullrich,A., Annu. Rev. Biochem. 57, 443-478 (1988)). Monomeric subclass Ireceptors have two cysteine-rich repeat sequences in the extracellulardomain; subclass II receptors have disulfide-linked heterotetrameric α₂β₂ -type structures with similar cysteine-rich repeat sequences; whereasthe extracellular domains of subclass III and IV receptors have five orthree immunoglobulin-like repeats, respectively. For example, receptorsfor insulin, epidermal growth factor (EGF), platelet-derived growthfactor (PDGF), insulin-like growth factor (IGF-1), colony-stimulatingfactor 1 (CSF-1), and hepatocyte growth factor (HGF) belong in thisfamily.

Because of their configuration, receptor tyrosine kinases can beenvisioned as membrane-associated allosteric enzymes. As in thesereceptors, in contrast to water-soluble enzymes, the ligand bindingdomain and the tyrosine kinase catalytic domain (protein tyrosine kinaseactivity) are separated by the plasma membrane, receptor activation dueto extracellular ligand binding must be translated across the membranebarrier in order to activate intracellular domain functions.

According to an allosteric receptor oligomerization model, ligandbinding and the resultant conformational alteration of the extracellulardomain induce receptor oligomerization, which, in turn, stabilizesinteractions between adjacent cytoplasmic domains and leads toactivation of kinase function by molecular interaction. Receptoroligomerization permits the transmission of a conformational change fromthe extracellular domain to the cytoplasmic domain without requiringalterations in the positioning of amino acid residues within thetransmembrane domain. The monomeric inactive receptors are inequilibrium with oligomeric activated receptors. The binding of growthfactors to their receptors stabilizes an oligomeric state whichpossesses enhanced ligand-binding affinity and elevated protein tyrosinekinase activity (Schlessinger, J., J. Cell Biol. 103, 2067-2072 (1986);Yarden, Y. and Schlessinger, J., Biochemistry 26, 1434-1442 (1987);Yarden, Y. and Schlessinger, J., Biochemistry 26, 1443-1451 (1987)). Amore general allosteric receptor oligomerization model is described inSchlessinger, a., Trends Biochem. Sci. 13, 443-447 (1988).

Receptor oligomerization which, for sake of simplicity, is commonlyillustrated by receptor dimerization, may be induced by monomericligands, such as EGF, that induce conformational changes resulting inreceptor-receptor interactions (Cochet, C. et al., J. Biol. Chem. 263,3290-3295 (1988)). Bivalent ligands, such as PDGF and CSF-1 mediatedimerization of neighboring receptors (Heldin, C. H. et al., J. Biol.Chem. 264, 8905-8912 (1989); Hammacher, A. et al., EMBO J. 8, 2489-2495(1989)).

The universality of this receptor activation model for all receptortyrosine kinases is supported by reports about the construction of fullyfunctional chimeric receptors consisting of major domains of differenttyrosine kinase receptor subclasses (Riedel, H. et al., EMBO J. 8,2943-2954 (1989)). Although in some cases heterodimer formation betweenstructurally very similar receptors (Hammacher, A. et al., supra for α-and β-type PDGF receptors; Soos, M. A. and Siddle, K., Biochem. J. 263,553-563 (1989) for insulin and IGF-1 receptors) has also beendemonstrated, direct proof that such hybrid receptors are indeedfunctional is not yet available. In general, more detailed analyses ofthe structural perturbations and requirements for ligand-inducedalterations in receptor tyrosine kinases has been hampered by thecomplexities of these membrane associated systems and by the lack ofsuitable quantities of highly purified natural or recombinant receptors.

For a general review of the signal transduction by receptors withtyrosine kinase activity see Ullrich, A. and Schlessinger, J., cell 81,203-212 (1990), and Bormann, B. J. and Engelman, D. M., Annu. Rev.Biophys. Biomol. Struct. 21, 223-266 (1992), and the references citedtherein.

A more recently discovered receptor tyrosine kinase is the HGF receptor(HGFr), which has been identified as the product of the c-METproto-oncogene (Bottaro et al., Science 251, 802-804 (1991); Naldini etal., Oncogene 6, 501-504 (1991)). MET was originally identified as atransforming gene in a chemically treated osteogenic sarcoma cell linethat had undergone a chromosomal translocation (Park, M. et al., Cell45, 895-904 (1986)). The mature HGFr is a disulfide linked heterodimerwhich arises by proteolytic processing of a glycosylated 190-kDaprecursor into a 50-kDa α-subunit and a 145-kDa β-subunit (Giordano, S.et al., Oncogene 4, 1383-1388 (1989)). The α-subunit is extracellularand the β-subunit contains an extracellular region, a singlemembrane-spanning domain and a tyrosine kinase domain. On normal cells,binding of HGF is required to activate the tyrosine kinase activity ofHGFr. The HGFr protein becomes phosphorylated on tyrosine residues ofthe 145-kDa β-subunit upon HGF binding.

Receptor oligomerization (dimerization) also appears to be critical forsignaling by certain cytokine receptors, particularly in a recentlydiscovered superfamily of single transmembrane receptors, designated asthe hematopoietin receptor superfamily (Bazan, et al., Biochem. Biophys.Res. Commun. 164, 788-795 (1989); D'Andrea, A. D., et al., Cell 58,1023-1024 (1989); Gearing, D. P. et al., EMBO J. 8, 3667-3676 (1989);Itoh, N. et al., Science 247, 324-327 (190); Idzerda, R. L. et al., J.Exp. Med. 171, 861-873 (1990); Godwin, R. G. et al., Cell 60, 941-951(1990); Fukunaga, R. et al., Cell 61, 341-350 (1990); Bazan, J. F. etal., Proc. Natl. Acad. Sci. USA 87, 6934-6938 (1990); Patthy, L., Cell61, 13-14 (1990); Abdel-Meguid, S. S. et al., Proc. Natl. Acad. Sci. USA84, 6434-6437 (1987); De Vos et al., Science 255, 306-312 (1992);Cosman, D. et al., Trends Biochem. Sci. 15, 265-270 (1990)). The membersof this superfamily include the receptors for growth hormone (GH),prolactin (PRL), placental lactogen (PL), and other cytokine andhematopoietic receptors, such as the receptors for interleukins 1 to 7(IL-1, IL-2, the β-subunit also known as p75, IL-3, IL-4, IL-5, IL-6,IL-7), erythropoietin (EPO), granolocyte colony stimulating factor(G-CSF), macrophage colony stimulating factor (M-CSF) andgranulocyte-macrophage colony stimulating factor (GM-CSF). Thesereceptors contain homologous extracellular ligand-binding domains andhighly variable intracellular domains that are not homologous to anyknown tyrosine kinase or other protein.

Recently Cunningham, B. C. et al., Science 254, 821-825 (1991) publishedevidence that dimerization is important for activation of hGH and othercytokine receptors. To analyze the structural requirements and mechanismfor hormone-induced changes in hGH, the authors used the extracellulardomain of the hGH receptor (hGH binding protein, hGHbp) produced in highyield by expression in E. coli. Results of crystallization, sizeexclusion chromatography, calorimetry studies and a fluorescencequenching assay showed that hGH forms a 1:2 complex with theextracellular domain of hGHbp. Based upon these and further studies itwas concluded that hGH contains two functionally distinct sites forbinding to two overlapping sites on the hGHbp in producing the hGH.(hGHbp)₂ complex, and that the formation of an analogous dimericreceptor complex on the cell surface is critical to the signaltransduction mechanism of hGH and probably homologous cytokinereceptors. The receptor dimerization mechanism was confirmed by thefinding that a hGH analog lacking the second receptor binding site (andtherefore unable to dimerize hGHbp) had decreased receptor bindingaffinity and decreased receptor down regulation to saturation.

Yet another example is the superfamily of nerve growth factor receptor(NGFR) related receptors, such as the tumor necrosis factor (TNF)receptors TNFR-I and TNFR-II, the Fas and Aps gene products, and severalT and B cell surface antigens. Currently included in this superfamilyare NGFR, found on neural cells, the B-cell antigen CD40, the MRC OX-40antigen, which is a marker of activated T cells of the CD4 phenotype,TNFR-I and TNFR-II which are found on a variety of cell types, a cDNA(4-1BB) which encodes a protein of unknown function and is obtained fromT-cell clones, and SFV-T2, an open reading frame in Shope fibroma virus.The members of this family are characterized by three or fourcysteine-rich motifs of about 40 amino acids in the extracellular domainof the molecule, and in some cases by a hinge-like region but no otherdomain types. Functionally, those members of this receptor superfamilythat have so far been characterized are usual in that they are able toreact with more than one ligand, and that these ligands are polymeric innature. It has been shown that the TNF receptors are activated byoligomerization because bivalent anti-TNFR antibodies but not monovalentantibody fragments (Fab fragments) were found to activate TNFR(Engelman, H. et al., J. Biol. Chem. 265, 14497-14504 (1990), and thereferences cited therein). It was suggested that a TNF-α trimer maytrigger signal transduction by cross-linking two cell surface TNFRmolecules (Ashkenazi, A. et al., Proc. Natl. Acad. Sci. USA 8810535-10539 (1991)). Similarly, the Fas and Aps gene products can beactivated by antibodies.

An object of the present invention is to provide methods forligand-induced receptor oligomerization.

It is another object to provide methods for making ligand variants thatact as competitive agonists of the corresponding native ligands.

It is a further object to provide methods for substantially recoveringligand biological activity lost as a result of a mutation.

It is a still further object to provide methods for converting ligandsthat are competitive antagonist of the action of their nativecounterparts into agonists.

It is yet another object to increase the half-life of ligands.

SUMMARY OF THE INVENTION

The present invention is based on observations obtained with a series ofrecombinant huHGF (rhuHGF) variants. The mature form of huHGF,corresponding to the major form purified from human serum, is adisulfide linked heterodimer derived by proteolytic cleavage of thehuman prohormone between amino acids R494 and V495. This cleavageprocess generates a molecule composed of an α-subunit of 440 amino acids(M_(r) 69 kDa) and a β-subunit of 234 amino acids (M_(r) 34 kDa). Thenucleotide sequence of the hHGF cDNA reveals that both the α- and theβ-chains are contained in a single open reading frame coding for apre-pro precursor protein. In the predicted primary structure of maturehHGF, an interchain S-S bridge is formed between Cys 487 of the α-chainand Cys 604 in the β-chain. The N-terminus of the α-chain is preceded by54 amino acids, starting with a methionine group. This segment includesa characteristic hydrophobic leader (signal) sequence of 31 residues andthe prosequence. The α-chain starts at amino acid (aa) 55, and containsfour Kringle domains. The Kringle 1 domain extends from about aa 128 toabout aa 206, the Kringle 2 domain is between about aa 211 and about aa288, the Kringle 3 domain is defined as extending from about aa 303 toabout aa 383, and the Kringle 4 domain extends from about aa 391 toabout aa 464 of the α-chain.

rhuHGF variants were produced to determine the structural and functionalimportance of the cleavage of the prohormone to the α/β dimer and of thekringle and protease-like domains. A series of C-terminal truncations ofhuHGF were made by deleting either the α-chain or the β-chain inaddition to a progressive number of kringle domains, and mutations wereintroduced at the one-chain to two-chain cleavage site, or within theprotease domain.

Some of the huHGF variants retained the ability to bind to theirreceptor (HGFr) with high affinity, but were defective in HGF biological(mitogenic) activity, and exhibited a reduced ability to inducephosphorylation of the HGFr.

It has been found that conformationally correct chimeric proteinscomprising the fusion of such variant HGF molecules to an immunoglobulinconstant domain sequence can be made, and that such chimeras retain theability to bind the HGFr.

In has further been found that the biological activity of HGF variantsthat were formerly capable of binding their receptor but lacked orexhibited substantially reduced HGF biological activity as compared towild-type huHGF could be substantially recovered in the form of HGFvariant-immunoglobulin chimeras.

Although the mechanism by which binding of HGF to HGFr activates theintracellular tyrosine kinase is not fully understood, it is believedthat receptor activation by the HGF variant-immunoglobulin chimerastested is due to the structural ability of the HGF-immunoglobulin heavychain dimers to induce receptor dimerization. HGF ligands coupled(oligomerized) by any other methods, e.g. via cysteine bridges, mayinduce receptor activation in an analogous manner.

Other receptors that require oligomerization for (full) biologicalactivity can also be activated by oligomerized (e.g. dimerized) ligandsequences, such as by chimeric molecules comprising receptor bindingdomain(s) from the corresponding native or variant ligands fused to animmunoglobulin constant domain sequence. Such receptors include otherreceptors with tyrosine kinase activity, such as receptors for insulin,EGF, PDGF, IGF-1, CSF-1; cytokines, e.g. members of the hematopoietinreceptor superfamily, such as hGH, hPRL, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, erythropoietin, G-CSF, M-CSF, and GM-CSF; and members of theNGFR superfamily, such as NGFR, TNFR-I, TNFR-II.

In one aspect, the present invention concerns a method for receptoractivation comprising (a) providing a conjugate comprising the directfusion of a first ligand and a second ligand capable of binding to firstand second receptors, respectively, wherein the first and secondreceptors are capable of oligomerization with each other, and areselected from the group consisting of receptors with tyrosine kinaseactivity, cytokine receptors, and members of the nerve growth factorreceptor superfamily, and (b) contacting the conjugate with the firstand second receptors whereby the first ligand binds to the firstreceptor and the second ligand binds to the second receptor.

In another aspect, the invention concerns a method for receptoractivation comprising (a) providing a conjugate comprising a firstligand and a second ligand capable of binding to first and secondreceptors, respectively, wherein the first and second receptors areselected from receptors with tyrosine kinase activity, and (b)contacting the conjugate with the first and second receptors whereby thefirst ligand binds to the first receptor and the second ligand binds tothe second receptor. In this embodiment, the first and second ligandsmay be directly fused to each other or may be connected by a covalentlinkage comprising a heterologous linker. The heterologous linker may,for example comprise an immunoglobulin constant and/or variable domainsequence, a moiety from a nonproteinaceous cross-linking agent, adisulfide bridge between the first and second ligands, or a polypeptidespacer sequence.

In a further aspect, the invention relates to a method for recoveringthe biological activity of a ligand variant capable of selective bindingto a receptor selected from the group consisting of receptors withtyrosine kinase activity, cytokine receptors, and members of the nervegrowth factor receptor superfamily, comprising:

a) directly fusing two molecules of the ligand variant to obtain ahomodimer; or

b) fusing the ligand variant to a second receptor binding amino acidsequence to obtain a heterodimer; and

c) contacting the homo- or heterodimer with the receptor such that onemolecule of the ligand variant binds to a first molecule of receptor anda second molecule of the ligand variant or the second receptor bindingsequence binds to a second molecule of the receptor.

If the receptor is from the family of receptor tyrosine kinases, the twoligand variants may be directly fused to each other, or, alternatively,may be connected by a heterologous linker.

In a still further aspect, the invention concerns a method for making anagonist for a native ligand of a receptor with tyrosine kinase activity,comprising dimerizing a first ligand variant capable of binding to thereceptor or coupling the first variant with a second ligand variantcapable of binding to the receptor.

In yet another aspect, the invention concerns a chimeric moleculecomprising a fusion of a first ligand capable of binding to a receptorwith tyrosine kinase activity to a first immunoglobulin constant domainsequence, and a fusion of a second ligand capable of binding to saidreceptor or to another receptor with tyrosine kinase activity to asecond immunoglobulin constant domain sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the α- and β-subunits of huHGF.Shown in the α-chain are the signal sequence (boxed region) whichencompasses amino acids 1-31, the predicted finger and four Kringledomains, each with their respective three disulfide bonds. The cleavagesite for generation of the heterodimeric α/β form of huHGF immediatelyfollows the P1 cleavage residue R494. This last residue has beenspecifically substituted with either E, D or A to generate HGFsingle-chain variants. The p-chain, which follows the cleavage site,contains homology to serine proteases. It is proposed that the α- andβ-chains are held together by a unique disulfide-bridge between C487(α)and C604(β) (Nakamura et al., 1989, supra). Three residues within theβ-chain have been substituted individually or in combination toreconstitute the authentic residues of a serine-protease. Schematicrepresentations of the mature forms of the C-terminal truncationvariants are depicted below: N-207, deleted after the first Kringle;N-303, deleted after the second Kringle; N-384, deleted after the thirdKringle and the α-chain. Also shown are the variants where deletions ofeach of the Kringles (ΔK1, ΔK2, ΔK3 and ΔK4) were introduced. In eachcase, the deletions specifically remove the entire Kringle from C1 toC6.

FIG. 2 shows the results of Western blot of wild-type rhuHGF andsingle-chain variants. Conditioned media from mock transfected 293 cellsor stable 293 cells expressing either wild-type rhuHGF (WT) or thevariants R494E, R494A or R494D were fractionated under reducingconditions on an 8% sodium-dodecyl sulfate-polyacrylamide gel andblotted. The blot was reacted with polyclonal anti-HGF antisera whichrecognizes epitopes primarily in the α-chain. Molecular masses(kilodaltons) of the marker are as indicated. Also indicated are thepositions of the α-chain and uncleaved single-chain forms of huHGF. Notethat the polyclonal antibody cross-reacts with an unidentified band (*)present even in the control transfected 293 cells, which do not expressdetectable quantities of huHGF.

FIGS. 3A and B: Mitogenic activity (A) and competitive receptor binding(B) of wild-type (WT) rhuHGF and single-chain variants. (A) Biologicalactivity was determined by the ability of WT rhuHGF and variants toinduce DNA synthesis of rat hepatocytes in primary culture as describedin Example 2. Shown are the mean cpm from duplicates in a representativeassay. Mock supernatant from control cells did not stimulate DNAsynthesis in these cells (no cpm increase above background levels). (B)To perform competitive binding, various dilutions of supernatants ofhuman 293 cells containing wt rhuHGF or variants were incubated with 50pM of the huHGF receptor-IgG fusion protein as described in Example 2.Data represent inhibition of binding as the percentage of any competingligand from a representative experiment and were corrected bysubtraction of background values from control 293 cells.

FIG. 4: Western blot of ligand-induced tyrosine-phosphorylation on the145kDa β-subunit of the HGF receptor by wild-type rhuHGF, single-chainor protease domain huHGF variants. Lysates from A549 cells incubated for5 minutes without (-) or with 200 ng/mL of purified wt rhuHGF (WT),single-chain (R494E) or double protease variants (Y673S,V692S) wereprepared and immunoprecipitated with an anti-HGF receptor antibody andblotted with anti-phosphotyrosine antibodies. Molecular masses(kilodaltons) are as indicated.

FIGS. 5A, B and C: Expression and proposed structures of HGF-IgGchimeras.

A. SDS-PAGE gel electrophoresis under reducing conditions.

B. SDS-PAGE gel electrophoresis under non-reducing conditions. Lane M:control (Mock) 293 cells; Lane 1: NK2-IgG; Lane 2: HGF-IgG; Lane 3:Y673S,V692S-IgG; Lane 4: R494E HGF-IgG.

C. Proposed structures of HGF variant-IgG chimeras.

FIGS. 6A and B: Competitive binding assay. Cell culture supernatants of293 cells expressing wild-type rhuHGF and various huHGF variant-IgGchimeras were tested for their ability to block the binding of CHO cellexpressed ¹²⁵ I rhuHGF to the extracellular domain of the human HGFrfused to the Fc constant region of human IgG-1, expressed and secretedfrom 293 cells.

FIGS. 7A, B and C: 3H-thymidine uptake assay. Conditioned media from 293cells expressing wild-type rhuHGF and various rhuHGF variant-IgGchimeras were tested for mitogenic effect in a 3H-thymidine uptakeassay. Mock: control 293 cells.

FIG. 8: Outline of expression plasmid pf-NK1. For bacterial expressionof HGF/NK1, an expression plasmid containing an alkaline phosphatasepromoter (phoA) adjacent to the coding sequence for the stII leaderpeptide to direct secretion of the expressed protein into theperiplasmic space was used. The coding sequence for the Flag epitope wasincluded to follow expression and purification steps. This Flag sequencewas followed by the coding sequence for mature HGF/NK1 (hatched segment)as indicated. The corresponding DNA and amino acid sequence of the stIIleader, Flag epitope (italics) and HGF/NK1 portions (N- and C-terminal,bold) are shown. The DNA sequences shown are SEQ ID NOS: 27, 28, and 29,which encode the amino acids sequences shown.

FIG. 9: Flow chart for purification of HGF/NK1 and fractionation of theheparin-sepharose pool by FPLC Mono S cation-exchange chromatography.Protein eluted from the heparin-sepharose column with a gradient of NaClwas dialyzed against 20 mM sodium-acetate, pH 6.0, 0.25M NaCl and aportion of this solution was loaded onto a FPLC Mono S cation-exchangecolumn equilibrated with the same buffer. A linear gradient from0.25M-1.5M NaCl was used to elute the bound protein, and fractions ofabout 1.5 ml were collected. The absorbance at 280 nM is shown.Fractions 6-9 were pooled and used in further experiments.

FIGS. 10A, B, C and D: SDS-PAGE analysis of crude bacterial extracts andpurified samples of HGF/NK1. Molecular masses (kilodaltons, kDa) of themarkers are indicated on the left of each gel, and the position ofmigration of HGF/NK1 is shown on the right. (A) Total cell lysates fromuninduced cells (lane 1) and from cells in which HGF/NK1 expression wasinduced (lanes 2-6) by phosphate starvation are shown. Lanes 2-6 showsamples from the fermentation run that were harvested at O.D. (550 nm)readings of 1.0; 7.2; 39; 66 and 72 respectively. Equal amounts ofprotein extract for each sample were fractionated under reducingcondition and detected with coomassie stain. (B) Western Blot analysisof crude lysates of E. coli 27C7 cells containing the HGF/NK1 expressionvector (lanes 1 and 3) or control 27C7 cells containing pBR322 alone(lanes 2 and 4) were analyzed under non-reducing and reducing conditionsas indicated. Proteins containing the FLAG epitope were detected asdescribed in Example 7. (C) FPLC Mono S chromatography fractions 7-11shown in lanes 1-5, respectively were analyzed under reducingconditions. Protein was detected by silver staining. (D) Proteinscontaining the Flag epitope present in FPLC fractions 1, 3, 5, 7, 9, 11,and 13 (lanes 1-7) were detected as in 10B.

FIGS. 11A and B: Competitive binding of (¹²⁵ I)-labelled HGF in thepresence of rhuHGF or HGF/NK1 to the purified soluble HGF receptor (A)or to the HGF receptor on A549 cells (B). Binding was performed in thepresence of 50 pM radioligand and the indicated concentrations of coldcompetitor. Shown are representative displacement curves of (¹²⁵ I)-HGFby unlabeled rhuHGF, HGF/NK1, and purified kringle 4 of plasminogen (K4plas) as indicated. Binding was performed in the presence of 100 pMradioligand and the indicated concentrations of cold competitor. Thedissociation constants (Kd) determined from three independentexperiments were 0.10±0.02 nM for rhu/HGF and 1.10±0.04 nM for HGF/NK1to the soluble HGF receptor (A), and 0.21±0.04 nM for rhuHGF and1.60±0.08 nM for HGF/NK1 (B) to the receptor on A549 cells.

FIG. 12: Western blot of ligand-induced phosphorylation on the 145 kDab-subunit of the HGF receptor by wild-type rhuHGF (A) and purifiedHGF/NK1 (B). Lysates from induced cells incubated for 15 min with theabove indicated factors were prepared and immunoprecipitated with ananti-receptor antibody. Western blots prepared from SDS-PAGE were probedwith anti-phosphotyrosine antibodies. Molecular masses (kDa) are asindicated.

FIGS. 13A and B: Effect of HGF/NK1 alone (A) or together with rhuHGF (B)on DNA synthesis of hepatocytes in primary culture. Hepatocytes wereexposed to increasing concentrations of rhu/HGF or HGF/NK1 alone (A) orincreasing concentrations of HGF/NK1 together with a fixed concentrationof HGF (0.64 nM corresponding to the amount of HGF required for 50%3H-Thymidine incorporation, B). Shown are representative curves fromthree independent experiments. As a control, purified kringle 4 ofplasminogen was tested for neutralizing HGF activity.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of the present invention the "receptor" can be anycell-surface receptor selected from receptors with tyrosine kinaseactivity, cytokine receptors and members of the nerve growth factorreceptor superfamily, the activation or signaling potential of which ismediated by oligomerization, irrespective of the actual mechanism bywhich the receptor oligomerization is induced, wherein "oligomerization"specifically includes dimerization as well as the formation of higheroligomers. The definition includes cell-surface receptors that arenormally activated a) by monomeric ligands (ligands with one receptorbinding domain), such as EGF, that induce conformational changes in theextracellular domain resulting in receptor-receptor interactions, b) bybivalent ligands (ligands with two receptor binding domains), such asPDGF, CSF-1, and hGH that mediate dimerization of neighboring receptors,or c) by interaction of the ligand with a disulfide stabilized receptordimer and subsequent intracomplex conformational change, such as insulinor IGF-1. Specifically covered by this definition are receptors withtyrosine kinase activity (receptor tyrosine kinases) and members of thehematopoietin and nerve growth factor receptor superfamilies.

"Cytokine" is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Included among the cytokines are growth hormone, insulin-like growthfactors, interleukins, hGH, N-methionyl hGH, bovine growth hormone,parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, glycoprotein hormones such as, follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH),hemopoietic growth factor, HGF, fibroblast growth factor, prolactin,placental lactogen, tumor necrosis factor-α and -β (TNF-α and -β),muellerian inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin, nerve growth factors, such as NGF-β, PDGF, transforminggrowth factors (TGFs) such as, TGF-α and TGF-β, insulin-like growthfactor-1 and -2 (IGF-1 and IGF-2), erythropoietin, osteoinductivefactors, interferons (IFNs) such as, IFN-α, IFN-βand IFN-γ, colonystimulating factors (CSFs) such as, M-CSF, GM-CSF, and G-CSF,interleukins (ILs) such as, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8 and other polypeptide factors. Cytokine receptors are receptorsbinding to cytokines as hereinabove defined.

The expressions "receptor with (protein) tyrosine kinase activity" and"receptor tyrosine kinase" and grammatical variants thereof, are usedinterchangeably and refer to receptors typically having a largeextracellular ligand binding domain, a single hydrophobic transmembraneregion and a tyrosine kinase catalytic domain, which can be classifiedinto subclasses (subclasses I-IV according to present knowledge) basedupon their sequence similarity and distinct structural characteristicsas defined by Schlessinger, J. (1988) Supra, and Ullrich, A. andSchlessinger, J. (1990),Supra. Among other highly conserved sequences ofunknown function, the tyrosine kinase domain of these receptors containsa consensus sequence Gly-X-Gly-X-X-Gly-X(15-20)Lys that functions aspart of the binding site for ATP. Receptor tyrosine kinases catalyze thephosphorylation of exogenous substrates as well as tyrosine residueswithin their own polypeptide chains. This family includes the insulinreceptor (insulin-R), epidermal growth factor receptor (EGF-R),platelet-derived growth factor receptors A and B (PDGF-R-A and -B),insulin-like growth factor receptor (IGF-1-R), cology-stimulating factor1 receptor (CSF-1-R), hepatocyte growth factor receptor (HGFr),HER2/neu, HER3/c-erbB-3, IRR, Xmrk and receptors for acidic fibroblastgrowth factor (FGF) and basic FGF, termed flg and bek. Theoligomerization mechanism implies the possible existence of hybridcomplexes between structurally very similar receptors such as PGDF-R-Aand -B, EGF-R and HER2/neu, or insulin-R and IGF-1-R (Hammacher et al.(1989), supra; Soos, M. A. and Siddle, K., Biochem. J. 263, 553-563(1989)).

EGF-R can serve as a model for subclass I receptor tyrosine kinasesactivated by a monovalent ligand. EGF-R is a single-chain polypeptide ofabout 170,000 kD composed of a large extracellular ligand bindingdomain, a single hydrophobic membrane spanning region, and a cytoplasmicregion with intrinsic protein tyrosine kinase activity (Ullrich, A. etal., Nature 309, 418-425 (1984)). Yarden and Schlessinger (Biochemistry26, 1434-1442 (1987); Biochemistry 24, 1443-1451 (1987)) demonstratedthat purified EGF-R undergoes rapid, reversible EGF-inducedoligomerization and that receptor oligomerization is an intrinsicproperty of the EGF-R. Similar results were obtained in living cells byCochet, C. et al., (J. Biol. Chem. 263, 3290-3295 (1988)). Based uponearlier structure-function studies and initial data from electronmicroscopic characterization of the purified extracellular domain of theEGF receptor, a four-domain model for the organization of theextracellular portion of the BGF receptor was proposed by Ullrich, A.and Schlessinger, J. (1990), supra. In this model, "domain III" and"domain I" are proposed to contribute most of the determinants thatenable the receptor to interact specifically with its ligand (EGF ortransforming growth factor-α-TGF-α), and it is suggested that theBGF-binding region lies in the cleft formed between domains III and I.HER2/neu (Lee, J. et al., EMBO J. 8, 167-173 (1989); Hazan, R. et al.,Cell. Growth Differ. 1, 3-7 (1990)), HER3/c-erbB-3 (Kraus, M. H. et al.,Proc. Natl. Acad. Sci. USA 86, 9193-9197 (1989)) and Xmrk (Wittbrodt, J.et al., Nature 341, 415-421 (1989)) belong in this subclass.

Typical representatives of the subclass II receptor tyrosine kinases arethe insulin-R and IGF-1-R (Ullrich et al., Nature 313, 756-761 (1985);Ullrich et al., EMBO J. 5, 2503-2512 (1986); Ebina, Y. et al., Cell 40,747-758 (1985); Perdue, J. F., Can. J. Biochem. Cell. Biol. 62,1237-1245 (1984); Rechler, M. M. and Nissley, S. P., Ann. Rev. Physiol.47, 425-442 (1985), and the references cited in these review articles;Lee et al., Mol. Endocrinol, 2, 404-422 (1988); Wilson et al., Mol.Endocrinol. 2, 1176-1185 (1988); Morgan et al., Nature 329, 3071-3072(1987)). Ligand binding to these receptors, which have aheterotetrameric structure (Lammer, R. et al., EMBO J. 8, 1369-1375(1989); Czech, M. Cell 59, 235-238 (1989)), induces allostericinteraction of two αβ halves within the disulfide bridge stabilizedreceptor complex (Ullrich, A. (1990), supra). This subclass alsoincludes IRR, a putative receptor for a ligand of the insulin family(Shier, P. and Watt, V. M., J. Biol. Chem. 264, 14605-14608 (1989)).

Subclass III receptor tyrosine kinases bind dimeric ligands that mediatedimerization of neighboring receptors. This subclass is represented byreceptors for PDGF-A and -B, and CSF-1. Human PGDF occurs as threeisoforms which are made up of disulfide-bonded A and B chains. Theisoforms bind to two different but structurally related cell surfacereceptors: PGDF-R-A and PGDF-R-B. The A-type receptor binds all threeisoforms (PGDF-AA, PGDF-AB, and PGDF-BB), whereas the B-type receptoronly binds PGDF-BB and PGDF-AB. It has been suggested that PDGF is abivalent ligand that activates its receptor by dimerization (Hammacher,A. et al., EMBO J. 8, 2489-2495 (1989)), and shown that dimerizationoccurs after ligand binding and is closely associated with receptorkinase activation (Heldin, C-H et al., J Biol. Chem. 264, 8905-8912(1989)) .

Subclass IV of the tyrosine kinase receptors includes the recentlydescribed receptors for acidic FGF (FGF-R flg) and basic FGF (FGF-R bek)(Ruta, M. et al., Proc. Natl. Acad. Sci. USA 86, 8722-8726 (1989) andPasquale, E. B. and Singer, S. J., Proc. Natl. Acad. Sci. USA 86,5449-5453 (1989), and references cited therein). These receptors exhibitthree related sequence repeats in their extracellular domains, and showweak but significant homology with the corresponding region of IL-1receptor.

The expression "hematopoietin receptor superfamily" is used to definesingle-pass transmembrane receptors, with a three-domain architecture:an extracellular domain hat binds the activating ligand, a shorttransmembrane segment, and a domain residing in the cytoplasm. Theextracellular domains of these receptors have low but significanthomology within their extracellular ligand-binding domain comprisingabout 200-210 amino acids. The homologous region is characterized byfour cysteine residues located in the N-terminal half of the region, anda Trp-Ser-X-Trp-Ser (WSXWS) motif located just outside themembrane-spanning domain. Further structural and functional details ofthese receptors are provided by Cosman, D. et al., (1990), supra. Thereceptors of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, prolactin,placental lactogen, growth hormone GM-CSF, G-CSF, M-CSF anderythropoietin have, for example, been identified as members of thisreceptor family.

IL-2-induced oligomerization of the IL-2 receptor was reported, forexample, by Ogura, T. et al., Mol. Biol. Med. 5, 123-13 (1988). Theyhave shown that high-affinity binding of IL-2 to its receptor gives riseto the formation of a ternary complex, comprising the IL-2 receptorα-subunit (p55), the β-subunit (p75, also referred to as the"converter"), and IL-2, by chemical crosslinking. The dimerization ofthe extracellular domain of hGH-R by a single hGH molecule was proposedto be relevant to the signal transduction mechanism for hGH receptor andother related cytokine receptors by Cunningham, B. C. et al., (1991),supra.

The expression "nerve growth factor receptor (NGFR) superfamily" is usedto describe a family of membrane proteins defined by the presence ofcysteine-rich motifs originally identified in the low-affinity NGFR.This superfamily, which was first described by Mallett, S. and Barclay,A. N., supra, includes two receptors for tumor necrosis factor (TNFR-Iand TNFR-II) and two lymphocyte proteins of so far undeterminedfunction.

The terms "first receptor" and "second receptor" as used throughout thespecification and the claims are used to designate receptors that arecapable of heterooligomer (heterodimer) formation in vivo as a result ofligand-induced receptor activation. Such receptors are usually locatedon similar (preferably identical) cell types, and may, but do not needto, exhibit structural homology. In a specific embodiment, the first andthe second receptors exhibit at least about 75% homology, and preferablyat least about 80%, more preferably at least about 85% homology in theiractive domains, and preferably have similar physiological functions. Ithas been mentioned before that hereto-receptor complexes might existbetween receptors such as PGDF-R-A and -B, EGF-R and HER2/neu, orinsulin-Rand IGF-1-R. Receptors for HGF and an HGF-like protein encodedby a gene recently identified on the DNF15S2 locus on human chromosome 3(3p21) (Han, S. et al., Biochemistry 30, 9768-9780 (1991)) are alsocandidates for heterodimer formation. Heterodimer formation between tworeceptors can be detected by standard methods of analytical chemistry,e.g. nondenaturing gel electrophoresis. In a preferred method, theinteraction of the receptors can be stabilized by utilizing a covalentcross-linking agent, essentially as described for EGF-R by Cochet, C. etal. (1988), supra, and the covalently linked, cross-linked receptors canbe analyzed by SDS gel-electrophoresis.

The term "ligand" is used to designate an organic molecule, or a peptideor polypeptide sequence capable of specific binding to a receptor ashereinabove defined. The definition includes any native ligand for areceptor or any region or derivative thereof retaining at least aqualitative receptor binding ability. Specifically excluded from thisdefinition are (agonist and antagonist) antibodies to a receptor andnoncovalent conjugates of an antibody and an antigen for that antibody.

In the molecules used in accordance with the present invention, thefirst and second ligands may be identical or different, and include twodifferent receptor binding domains from a native bivalent ligand, or atleast the receptor binding domains from two identical or differentligands for the same or two different receptors, and derivatives of suchnative receptor binding sequences. It has been proposed that hybridcomplexes might exist between structurally and/or functionally similarreceptors as part of the oligomerization activation mechanism. Suchreceptors are, for example, the A-type and B-type PDGF receptors, theEGF-R and HER2/neu, the insulin-R and IGF-1-R. In some cases.heterodimer formation has already been demonstrated (Hammacher et al.(1989), supra; Soos and Siddle (1989), supra). Molecules comprising thereceptor binding domains of ligands for such closely related receptorsare specifically within the scope herein.

The term "derivative" is used to define amino acid sequence andglycosylation variants, and covalent modifications of a native ligand.

The term "variant" is used to define amino acid sequence andglycosylation variants of a native ligand.

The terms "native ligand" and "wild-type ligand" are usedinterchangeably and refer to a ligand amino acid sequence as occurringin nature ("native sequence ligand"), including mature, pre-pro and proforms of such ligands, purified from natural source, chemicallysynthesized or recombinantly produced. It will be understood thatnatural allelic variations exist and can occur among individuals, asdemonstrated by one or more amino acid differences in the amino acidsequence of each individual. These allelic variations are specificallywithin the scope herein.

The terms "amino acid" and "amino acids" refer to all naturallyoccurring L-α-amino acids. The amino acids are identified by either thesingle-letter or three-letter designations:

    ______________________________________                                        Asp  D        aspartic acid                                                                             Ile  I     isoleucine                               Thr  T        threonine   Leu  L     leucine                                  Ser  S        serine      Tyr  Y     tyrosine                                 Glu  E        glutamic acid                                                                             Phe  F     phenylalanine                            Pro  P        proline     His  H     histidine                                Gly  G        glycine     Lys  K     lysine                                   Ala  A        alanine     Arg  R     arginine                                 Cys  C        cysteine    Trp  W     tryptophan                               Val  V        valine      Gln  Q     glutamine                                Met  M        methionine  Asn  N     asparagine                               ______________________________________                                    

These amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residue: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

The term "amino acid sequence variant" refers to molecules with somedifferences in their amino acid sequences as compared to a nativesequence of a ligand. Ordinarily, the amino acid sequence variants willpossess at least about 70% homology with at least one receptor bindingdomain of a native ligand, and preferably, they will be at least about80%, more preferably at least about 90% homologous with a receptorbinding domain of a native ligand. The amino acid sequence variantspossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence of a native ligand.

"Homology" is defined as the percentage of residues in the candidateamino acid sequence that are identical with the residues in the aminoacid sequence of a receptor binding domain of a native ligand afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent homology. Methods and computer programs for thealignment are well known in the art.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative ligand sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

Deletional variants are those with one or more amino acids in the nativeligand amino acid sequence removed. Ordinarily, deletional variants willhave one or two amino acids deleted in a particular region of themolecule.

The term "glycosylation variant" is used to refer to a ligand having aglycosylation profile different from that of a native ligand.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to theside-chain of an asparagine residue. The tripeptide sequences,asparagine-X-serine and asparagine-X-threonine, wherein X is any aminoacid except proline, are recognition sequences for enzymatic attachmentof the carbohydrate moiety to the asparagine side chain. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be involved in O-linked glycosylation. Anydifference in the location and/or nature of the carbohydrate moietiespresent in a ligand as compared to its native counterpart is within thescope herein.

The glycosylation pattern of native ligands can be determined by wellknown techniques of analytical chemistry, including HPAE chromatography(Hardy, M. R. et al., Anal. Biochem. 170, 54-62 (1988)), methylationanalysis to determine glycosyl-linkage composition (Lindberg, B., Meth.Enzymol. 28. 178-195 (1972); Waeghe, T. J. et al., Carbohydr. Res. 123,281-304 (1983)), NMR spectroscopy, mass spectrometry, etc.

For ease, changes in the glycosylation pattern of a native ligand areusually made at the DNA level, essentially using the techniquesdiscussed hereinabove with respect to the amino acid sequence variants.

Chemical or enzymatic coupling of glycosydes to the ligands of thepresent invention may also be used to modify or increase the number orprofile of carbohydrate substituents. These procedures are advantageousin that they do not require production of the polypeptide that iscapable of O-linked (or N-linked) glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free hydroxyl groups such asthose of cysteine, (d) free sulfhydryl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan or (f) the amide group ofglutamine. These methods are described in WO 87/05330 (published 11 Sep.1987), and in Aplin and Wriston, CRC Crit. Rev, Biochem., pp. 259-306

Carbohydrate moieties present on a ligand may also be removed chemicallyor enzymatically. Chemical deglycosylation requires exposure totrifluoromethanesulfonic acid or an equivalent compound. This treatmentresults in the cleavage of most or all sugars, except the linking sugar,while leaving the polypeptide intact. Chemical deglycosylation isdescribed by Hakimuddin et al., Arch. Biochem. Biophys. 259, 52 (1987)and by Edge et al., Anal. Biochem. 118, 131 (1981). Carbohydratemoieties can be removed by a variety of endo- and exoglycosidases asdescribed by Thotakura et al., Meth. Enzymol. 138, 350 (1987).Glycosylation is suppressed by tunicamycin as described by Duskin etal., J. Biol. Chem. 257, 3105 (1982). Tunicamycin blocks the formationof protein-N-glycosydase linkages.

Glycosylation variants of the ligands herein can also be produced byselecting appropriate host cells. Yeast, for example, introduceglycosylation which varies significantly from that of mammalian systems.Similarly, mammalian cells having a different species (e.g. hamster,murine, insect, porcine, bovine or ovine) or tissue (e.g. lung, liver,lymphoid, mesenchymal or epidermal) origin than the source of theligand, are routinely screened for the ability to introduce variantglycosylation.

The terms "ligand variant" and "variant ligand", that are usedinterchangeably, include both amino acid sequence variants andglycosylation variants of a native ligand.

"Covalent derivatives" include modifications of a native ligand with anorganic proteinaceous or non-proteinaceous derivatizing agent, andpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theligand with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-ligandantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation. Certainpost-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the ligands used in accordance with thepresent invention. Other post-translational modifications includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the α-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W. H. Freeman & Co., San Francisco,pp. 79-86 (1983)).

Covalent derivatives specifically include fusion molecules in whichligands of the invention ar covalently bonded to a nonproteinaceouspolymer. The nonproteinaceous polymer ordinarily is a hydrophilicsynthetic polymer, i.e. a polymer not otherwise found in nature.However, polymers which exist in nature and are produced by recombinantor in vitro methods are useful, as are polymers which are isolated fromnature. Hydrophilic polyvinyl polymers fall within the scope of thisinvention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularlyuseful are polyvinylalkylene ethers such a polyethylene glycol,polypropylene glycol.

The ligands may be linked to various nonproteinaceous polymers, such aspolyethylene glycol, polypropylene glycol or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Native ligands and derivatives that can activate receptors in accordancewith the present invention are well known in the art or can be preparedby art known methods.

The operability of the present invention was first demonstrated withHGFr/HGF or HGF variant receptor/ligand pairs. HGF was identifiedinitially as a mitogen for hepatocytes (Michalopoulos et al., CancerRes. 44, 4414-4419 (1984); Russel et al., J. Cell. Physiol. 119, 183-192(1984) and Nakamura et al., Biochem. Biophys. Res. Comm. 122:1450-1459(1984)). Nakamura et al., Supra reported the purification of HGF fromthe serum of partially hepatectomized rats. Subsequently, HGF waspurified from rat platelets, and its subunit structure was determined(Nakamura et al., Proc. Natl. Acad. Sci. USA, 83, 6489-6493 (1986); andNakamura et al., FEBS Letters 242, 311-316 (1987)). The purification ofhuman HGF (huHGF) from human plasma was first described by Gohda et al.,J. Clin. Invest. 81, 414-419 (1988).

Both rat HGF and huHGF have been molecularly cloned, including thecloning and sequencing of a naturally occurring variant lacking 5 aminoacids designated "delta5 HGF" (Miyazawa et al., Biochem. Biophys. Res.Comm. 163, 967-973 (1989); Nakamura et al., Nature 342, 440-443 (1989);Seki et al, Biochem. and Biophys. Res. Commun. 172, 321-327 (1990);Tashiro et al., Proc. Natl. Acad. Sci. USA 87, 3200-3204 (1990); Okajimaet al., Eur. J. Biochem. 193, 375-381 (1990)).

The mature form of huHGF, corresponding to the major form purified fromhuman serum, is a disulfide linked heterodimer derived by proteolyticcleavage of the human pro-hormone between amino acids R494 and V495.This cleavage process generates a molecule composed of an α-subunit of440 amino acids (M_(r) 69 kDa) and a β-subunit of 234 amino acids (M_(r)34 kDa) . The nucleotide sequence of the hHGF cDNA reveals that both theα- and the β-chains are contained in a single open reading frame codingfor a pre-pro precursor protein. In the predicted primary structure ofmature hHGF, an interchain S-S bridge is formed between Cys 487 of theα-chain and Cys 604 in the β-chain (see Nakamura et al., Nature, supra).The N-terminus of the α-chain is preceded by 54 amino acids, startingwith a methionine group. This segment includes a characteristichydrophobic leader (signal) sequence of 31 residues and the prosequence.The α-chain starts at amino acid (aa) 55, and contains four Kringledomains. The Kringle 1 domain extends from about aa 128 to about aa 206,the Kringle 2 domain is between about aa 211 and about aa 288, theKringle 3 domain is defined as extending from about aa 303 to about aa383, and the Kringle 4 domain extends from about aa 391 to about aa 464of the α-chain. It will be understood that the definition of the variousKringle domains is based on their homology with kringle-like domains ofother proteins (prothrombin, plasminogen), therefore, the above limitsare only approximate. As yet, the function of these Kringles has notbeen determined. The β-chain of huHGF shows high homology to thecatalytic domain of serine proteases (38% homology to the plasminogenserine protease domain). However, two of the three residues which formthe catalytic triad of serine proteases are not conserved in huHGF.Therefore, despite its serine protease-like domain, hHGF appears to haveno proteolytic activity and the precise role of the β-chain remainsunknown. HGF contains four putative glycosylation sites, which arelocated at positions 294 and 402 of the α-chain and at positions 566 and653 of the β-chain.

In a portion of cDNA isolated from human leukocytes in-frame deletion of15 base pairs was observed. Transient expression of the cDNA sequence inCOS-1 cells revealed that the encoded HGF molecule (delta5 HGF) lacking5 amino acids in the Kringle 1 domain was fully functional (Seki et al.,supra).

A naturally occurring huHGF variant has recently been identified whichcorresponds to an alternative spliced form of the huHGF transcriptcontaining the coding sequences for the N-terminal finger and first twokringle domains of mature huHGF (Chan et al., Science 254, 1382-1385(1991); Miyazawa et al., Eur. J. Biochem. 197, 15-22 (1991)). Thisvariant, designated HGF/NK2, has been proposed to be a competitiveantagonist of mature huHGF.

The comparison of the amino acid sequence of rat HGF with that of huHGFrevealed that the two sequences are highly conserved and have the samecharacteristic structural features. The length of the four Kringledomains in rat HGF is exactly the same as in huHGF. Furthermore, thecysteine residues are located in exactly the same positions; anindication of similar three-dimensional structures (Okajima et al.,supra; Tashiro et al., supra).

As used herein, the terms "hepatocyte growth factor", "HGF" and "huHGF"refer to a (human) growth factor capable of specific binding to areceptor of wild-type (human) HGF, which growth factor typically has astructure with six domains (finger, Kringle 1, Kringle 2, Kringle 3,Kringle 4 and serine protease domains), but nonetheless may have fewerdomains or may have some of its domains repeated if it still retains itsqualitative HGF receptor binding ability. This definition specificallyincludes the delta5 huHGF as disclosed by Seki et al., supra. The terms"hepatocyte growth factor" and "HGF" also include hepatocyte growthfactor from any non-human animal species, and in particular rat HGF.

The terms "wild-type human hepatocyte growth factor", "native humanhepatocyte growth factor", "wild-type (wt) huHGF", and "native huHGF"refer to native sequence human HGF, i.e., that encoded by the cDNAsequence published by Miyazawa, et al. 1989, supra, or Nakamura et al.,1989, supra, including its mature, pre, pre-pro, and pro forms, purifiedfrom natural source, chemically synthesized or recombinantly produced.The sequences reported by Miyazawa et al. and Nakamura et al, differ in14 amino acids. The reason for the differences is not entirely clear;polymorphism or cloning artifacts are among the possibilities. Bothsequences are specifically encompassed by the foregoing terms as definedfor the purpose of the present invention. It will be understood thatnatural allelic variations exist and can occur among individuals, asdemonstrated by one or more amino acid differences in the amino acidsequence of each individual. Amino acid positions in the variant huHGFmolecules herein are indicated in accordance with the numbering ofMiyazawa et al. 1989, supra.

In the course of a recent study of the structure-activity andstructure-receptor binding relationship in amino acid sequence variantsof HGF,.the results of which are disclosed in the examples hereinafter,domains critical for ligand binding and/or activation have beenidentified in the wild-type HGF amino acid sequence. A number ofC-terminal truncations of HGF were made by deleting either the β-chainor the β-chain in addition to a progressive number of kringles. Deletionof the first Kringle (variant ΔK1) of HGF affected biological activitymost, showing at least a 100-fold reduction (SA<0.2% of wt rhuHGF).Similarly, binding of this variant was also affected as it failed tocompete for binding with wt rhuHGF. Deletion of all other Kringles(variants ΔK2, ΔK3 or ΔK4) also induces severely reduced mitogenicactivity. However, the receptor binding affinities (Kds) of thesedeletion variants remained close to that observed with wt rhuHGF. Thesedata showed that kringles K3 and K4 are not required for receptorbinding, and were in agreement with previous observations by Miyazawa etal., 1991 supra and Chan et al., 1991 supra, in the sense that variantN-303, which in amino acid sequence is very similar to HGF/NK2, retainsthe ability to compete efficiently for binding to the HGF receptor(Kd˜280 pM). Furthermore, the observations that N-303 is sufficient bindto the receptor and that the second kringle is not required for bindingthe HGF receptor (in the context of the remainder of the molecule)suggest that the receptor binding domain is contained within the fingerand first kringle of huHGF.

To elucidate the functional importance of the protease domain of HGF,several single, double and triple mutations were made in order toreconstitute a potential serine-protease active site. The amino acidsubstitutions were made at positions 534, 673 and 692 of the wild-typehHGF amino acid sequence. In most cases, the biological activity wassubstantially reduced without substantial decrease in the ligand bindingaffinity. The biological activity of the double variants Q534H,Y673S andY673S,V692S and of the triple variant Q534H,Y673S,V692S were less than3% compared to WT rhuHGF. However, the Kd of these variants was notsignificantly different from that of the wild-type human HGF molecule.These results indicate that certain mutations within the β-chain of HGFblock mitogenic activity but have no significant effect on the receptorbinding ability of HGF. Thus, it appears that these mutants aredefective in an activity subsequent to receptor binding.

Alterations that potentially increase the receptor binding capacity ofHGF are, for example, in the amino acid region corresponding to apotential serine protease active site. This region includes amino acidsQ534, Y673 and V692 in the wild-type huHGF amino acid sequence. Thereplacement of these amino acids with any other amino acid, andpreferably with amino acids of different size and/or polarity isbelieved to further improve the receptor binding properties of the HGFvariant.

Additional alterations may be at the C-terminal end and/or in theKringle domains of the HGF molecule. In addition to the deletion mutantsreferred to hereinabove, HGF variants with alterations within theKringle 1 domain are of great interest. As we have found that thereceptor binding domain is contained within the finger and the Kringle 1regions of the HGF molecule, amino acid alterations within these domainsare expected to significantly alter the receptor binding properties (andthe biological activity) of the variants of the present invention.Alterations at residues that are most exposed to the interior in theKringle structure (mostly charged residues) are particularly likely tocause profound changes in the receptor binding properties and/orbiological activity of the HGF variants.

Further ligands for receptors with tyrosine kinase activity arecommercially available (e.g. insulin) and/or are characterized by theirnucleotide and deduced amino acid sequences. Their biological activitiesare also known.

EGF and related proteins are known (Carpenter, G. and Cohen, S. Ann.Rev. Biochem. 48, 193-216 (1979); Massanque, J., J. Biol. Chem. 255,21393-21396 (1990); Gray, A. et al., Nature 303, 722-725 (1983); Bell,G. I. et al., Nucl. Acid. Res. 14, 8427-8446 (1986)).

The amino acids sequence and preparation of human insulin-like growthfactors 1 and 2 (IGF-I and IGF-II) is, for example, disclosed in EP128,733 (published 19 Dec. 1984).

Ligands for HER2/neu (p185^(HER2)) have been designated as "heregulin-2"(HRG2) polypeptides, and include HRG2-α and HRG2-β1, -β2and -β3. Thestructure, preparation and use of these ligands and their derivatives,including amino acid sequence variants, are disclosed in copending U.S.applications Ser. Nos. 07/705,256 (filed 24 May 1991); 07/790,801 (filed8 Nov. 1991); and 07/880,917 (filed 11 May 1992). The amino acidsequence of HRG shares a number a features with the EGF family oftransmembrane bound growth factors. Alignment of the amino acidsequences in the region of the BGF motif and flanking transmembranedomain of several human EGF related proteins shows a relatively greatdegree of homology with heparin binding EGF-like growth factor (HB-EGF)(Higashiyama et al., Science 251, 936-939 (1991); amphiregulin (AR))(Plowman, G. D. et al., Mol. Cell. Biol. 10. 1969-1981 (1990));transforming growth factor-α (TGF-α); EGF (Bell, G. I. et al. (1986),supra); and schwanoma-derived growth factor (Kimura, H. et al., 3489,257-260 (1990)).

A typical representative of bivalent ligands for receptors with tyrosinekinase activity is platelet derived growth factor (PDGF). PDGF is amajor mitogen in serum for connective tissue-derived cells in culture(see Ross, R. et al., Cell 46, 155-169 (1986) for review). It is a 30-kDdimer composed of disulfide-bonded A and B polypeptide chains. All threepossible isoforms of the two chains, PDGF-AA, PDGF-AB, and PDGF-BB, havebeen identified and purified from natural sources (Heldin, C. -H. etal., Nature 319, 511-514 (1986); Hammacher, A. et al., Eur. J. Biochem.176, 1790186 (1988); Stroobant, P. and Waterfield, M. D., EMBO J. 3,2963-2967 (1984)). The different isoforms have been found to differ infunctional activities, most likely due to different bindingspecificities to two separate receptor classes (Nister, M. et al., Cell52, 791-799 (1988); Heldin, C. -H. et al., EMBO J. 7, 1387-1393 (1988);Hart, C. E. et al., Science 240, 1529-1531 (1988)). The A-type PDGFreceptor binds all three isoforms f PDGF, whereas the B-type receptorbinds PDGF-BB with high affinity and PDGF-AB with lower affinity butdoes not bind PDGF-AA with any appreciable affinity.

Another bivalent ligand is human growth hormone (hGH). hGH is a memberof an homologous hormone family that includes placental lactogens,prolactins, and other genetic and species variants of growth hormone,and is usually referred to as the family of hematopoietins, includingpituitary and hematopoietic hormones (Nicoll, C. S. et al., EndocrineReviews 7, 169 (1986)). The cloned gene for hGH has been expressed in asecreted form in E. coli (Chang, C. N. et al., Gene 55, 189 (1987)), andits nucleotide and amino acid sequences have been reported (Goeddel etal., Nature 281, 544 (1979); Gray et al., Gene 39, 247 (1985)). Thethree-dimensional folding pattern of porcine growth hormone (pGH) hasbeen reported (Abdel-Meguid, S. S. et al., Proc. Natl. Acad. Sci. USA 846434 (1987)). hGH receptor and antibody binding sites have beenidentified by homolog-scanning mutagenesis (Cunningham, B. et al.,Science 243, 1330 (1989)). GH variant with N-terminal truncations orwith mutations in the N-terminal region are known (Gertler et al.,Endocrinology 118, 720 (1986); Ashkenazi, A. et al., Endocrinology 121,414 (1987); and Binder, Mol. Endo. 7, 1060-1068 (1990)). Antagonistvariants of hGH were described by Chan et al., Mol. Endo. 5, 1845 (1991)and in the references cited therein, and in WO 91/05853. hGH variantsare also disclosed by Cunningham et al., Science 244, 1081 (1989); andScience 243, 1330-1336 (1989).

The structures of several other hematopoietic ligands have beendetermined recently. Granulocyte-macrophage colony stimulating factor(GM-CSF) and IL-4 are about 60 residues shorter than growth hormone.Both the crystal structure of GM-CSF (Diederichs, K. et al., Science254, 1779-1782 (1991); Walter, M. R. et al., J. Mol. Biol. 224,1075-1085 (1992)), and the NMR structure of IL-4 (Powers, R. et al.,Science 256, 1673-1677 (1992); Smith, L. J. et al., J. Mol. Biol. 224,899-904 (1992)) reveal the same topology as GH, but with an additionalstructural motif not seen before: a short segment of B-ribbon formed byresidues in the long crossover connections. From the evidence thus faravailable, it appears that two topologically-conserved receptor-bindingsites are a common theme throughout the hematopoietins. Whereas nativehGH use these two sites to bind two copies of the same receptor, in manyother cases such as IL-2, IL-3, GM-CSF and others, the equivalentsegments may form binding interfaces for two different receptorsubunits.

Receptor binding domains in a native ligand sequence can be determinedby methods known in the art, including X-ray studies, mutationalanalyses, and antibody binding studies. The mutational approachesinclude the techniques of random saturation mutagenesis coupled withselection of escape mutants, insertional mutagenesis, andhomolog-scanning mutagenesis (replacement of sequences from humanligands, which bind the corresponding receptor, with unconservedsequences of a corresponding ligand from another animal species, e.g.mouse, which do not bind the human receptor) . Another strategy suitablefor identifying receptor-binding domains in ligands is known asalanine-scanning mutagenesis (ALA-scan, Cunningham and Wells, Science244, 1081-1985 (1989)). This method involves the identification ofregions that contain charged amino acid side chains. The chargedresidues in each region identified (i.e. Arg, Asp, His, Lys, and Glu)are replaced (one region per mutant molecule) with alanine and thereceptor binding of the obtained ligands is tested, to assess theimportance of the particular region in receptor binding. Another methodfor identifying active domains in polypeptides along with a number ofhGH variants is disclosed in WO 90/04788 (published 3 May 1990).According to this method, the active domains (e.g. receptor bindingdomains) in a polypeptide are determined by substituting selected aminoacid segments of the polypeptide with an analogous polypeptide segmentfrom an analog of the polypeptide which has a different activity withthe target substance (e.g. receptor) as compared to the parentpolypeptide. A further powerful method for the localization of receptorbinding domain(s) in a ligand is through the use of neutralizing(blocking) monoclonal antibodies (MAbs). Usually a combination of theseand similar methods is used for localizing the domains important forreceptor binding.

Derivatives, such as amino acid sequence variants, of the foregoing andother ligands for receptors that require oligomerization for activationof receptor function are also known or can be easily prepared by methodsknown in the art, such as by site directed mutagenesis of the DNAencoding the precursor or parental ligand, thereby producing DNAencoding the variant. Modifications of the DNA encoding the variantligand molecules must not place the sequence out of reading frame, andpreferably will not create complementary regions which could producesecondary mRNA structure. The DNA encoding the variant ligand isinserted into an appropriate expression vector, and suitable host cellsare then transfected with this DNA. Culturing the host cells in anappropriate medium will result in the production of polypeptides encodedby the DNA, and secretion of the polypeptide into the host cell culturemedium. These techniques will be described in more detail hereinbelow.

Alternatively, amino acid variants of native ligand molecules areprepared by in vitro synthesis using standard solid-phase peptidesynthesis procedures as described by Merrifield (J. Am. Chem. Soc.85:2149 (1963), although other equivalent chemical syntheses known inthe art may be used. Solid-phase synthesis is initiated from theC-terminus of the peptide by coupling a protected α-amino acid to asuitable resin. The amino acids are coupled the peptide chain usingtechniques well known in the art for the formation of peptide bonds.

Glycosylation variants of native ligand molecules may be prepared bytechniques known in the art. Chemical and enzymatic coupling ofglycosides to proteins can be accomplished using a variety of activatedgroups, for example, as described by Alpin and Wriston in CRC Crit. Rev.Biochem. pp. 259-306 (1981). The advantages of the chemical couplingtechniques are that they are relatively simple and do not need thecomplicated enzymatic machinery required for natural O- and N-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups such asthose of glutamic acid and aspartic acid, (c) free sulfhydryl groupssuch as those of cysteine, (d) free hydroxyl groups such as those ofserine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan or (f) the amide groupof glutamine. These methods are described in WO 87/05330 (published 11Sep. 1987). Carbohydrates present in a native ligand molecule can, forexample, be removed by the use of an endoglycosidase, such asEndoglycosydase H (Endo-H), which is capable of (partial) removal ofhigh mannose and hybrid oligosaccharides. This treatment is accomplishedvia techniques known per se, for example, according to the method ofTarentino et al., J. Biol. Chem. 249, 811 (1974), Trimble et al., Anal.Biochem. 141, 515 (1984) and Little et al., Biochem. 23, 6191 (1984).More preferably, glycosylation variants of ligands are made byappropriate mutations at the DNA level, to provide a protein with thedesired, altered glycosylation pattern.

In accordance with the present invention, a first and a second ligand(which may be identical or different) may be directly fused to eachother. Such fusion molecules can be prepared by expression of theencoding DNA sequence in a suitable microorganism or cell culture,employing standard techniques of recombinant DNA technology.Alternatively, they may be obtained by chemical synthesis.

The term "heterologous linker" is used to refer to any organic orinorganic linker molecules coupling two ligands (as hereinabovedefined), provided that they are different from a linker connecting thetwo ligands in their native environment, e.g. in a bivalent ligand.Should the two ligands be connected in their native environment with anamino acid sequence, variants encoded by a DNA sequence capable ofhybridizing under stringent conditions with the DNA sequence encodingsuch connecting amino acid sequence are specifically excluded from thedefinition of the term "heterologous linker".

"Stringent conditions" are overnight incubation at 37° C. in a solutioncomprising: 40% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextrane sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 1× SSC at about 50° C.

In a preferred embodiment, the linker comprises an immunoglobulinsequence.

The term "immunoglobulin" generally refers to polypeptides comprising alight or heavy chain usually both disulfide bonded in the native "Y"configuration, although other linkage between them, including tetramersor aggregates thereof, is within the scope hereof.

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982);EP 120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Kohler etal., Proc. Nat'l. Acad. Sci. USA 77:2197 (1980); Raso et al., CancerRes. 41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984);Morrison, Science 229:1202 (1985); Morrison et al., Proc. Nat'l. Acad.Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559.Reassorted immunoglobulin chains also are known. See for example U.S.Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references citedtherein. The immunoglobulin moiety in the chimeras of the presentinvention may be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes,IgA, IgE, IgD or IgM, but preferably IgG-1 or IgG-3.

Chimeras constructed from a receptor sequence linked to an appropriateimmunoglobulin constant domain sequence (immunoadhesins) are known inthe art. Immunodhesins reported in the literature include fusions of theT cell receptor* (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84,2936-2940 (1987)); CD4* (Capon et al., Nature 337, 525-531 (1989);Traunecker et al., Nature 339, 68-70 (1989); Zettmeissl et al., DNA CellBiol. USA, 9, 347-353 (1990); Byrn et al., Nature 344, 667-670 (1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110,2221-2229 (1990); Watson et al., Nature 349, 164-167 (1991)); CD44*(Aruffo et al., Cell 61, 1303-1313 (1990)); CD28* and B7* (Linsley etal., J. Exp. Med. 173, 721-730 (1991)); CTLA-4* (Lisley et al., J. Expo.Med. 174, 561-569 (1991)); CD22* (Stamenkovic et al., Cell 66. 1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88,10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27, 2883-2886(1991); Peppel et al., J. Exp. Med. 174, 1483-1489 (1991)); NP receptors(Bennett et al., J. Biol. Chem. 266, 23060-23067 (1991)); IgE receptorα-chain* (Ridgway and Gorman, J. Cell Biol. 115, abstr. 1448 (1991));HGF receptor (Mark, M. R. et al., 1992, J. Biol. Chem. submitted), wherethe asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily. These immunoadhesins were manufactured withdifferent goals in mind, they are, however, all common in that they canpossess many of the desired chemical and biological properties of humanantibodies.

Ligand-immunoglobulin chimeras are disclosed in copending applicationsSer. Nos. 07/834,902, now U.S. Pat. No. 5,304,640; filed 13, Feb. 1992(for L-selection ligands); 07/884,811, now U.S. Pat. No. 5,316,921; and07/885,971 , now U.S. Pat. No. 5,328,837; both filed 18 May 1992 (forHGF variants). These chimeras can be made in a similar way to theconstruction of receptor-immunoglobulin chimeras.

Ordinarily, the ligand is fused C-terminally to the N-terminus of theconstant region of an immunoglobulin in place of the variable region(s),however N-terminal fusions of the selectin variants are also desirable.

Typically, such fusions retain at least functionally active hinge, CH2and CH3 domains of the constant region of an immunoglobulin heavy chain.Fusions are also made to the C-terminus of the Fc portion of a constantdomain, or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. This ordinarily is accomplishedby constructing the appropriate DNA sequence and expressing it inrecombinant cell culture. Alternatively, however, theligand-immunoglobulin chimeras of this invention may be synthesizedaccording to known methods.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of theligand-immunoglobulin chimeras.

In some embodiments, the hybrid immunoglobulins are assembled asmonomers, or hereto- or homo-multimers, and particularly as dimers ortetramers, essentially as illustrated in WO 91/08298.

In a preferred embodiment, the C-terminus of a ligand sequence whichcontains the binding site(s) for a receptor, is fused to the N-terminusof the C-terminal portion of an antibody (in particular the Fc domain),containing the effector functions of an immunoglobulin, e.g.immunoglobulin G₁ (IgG-1). It is possible to fuse the entire heavy chainconstant region to the sequence containing the receptor binding site(s).However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site (which defines IgG Fc chemically;residue 216, taking the first residue of heavy chain constant region tobe 114 (Kobet et al., supra), or analogous sites of otherimmunoglobulins) is used in the fusion. In a particularly preferredembodiment, the amino acid sequence containing the receptor bindingsite(s) is fused to the hinge region and CH2 and CH3 or CH1, hinge, CH2and CH3 domains of an IgG-1, IgG-2, or IgG-3 heavy chain. The precisesite at which the fusion is made is not critical, and the optimal sitecan be determined by routine experimentation.

In some embodiments, the ligand-immunoglobulin chimeras are assembled ashetero-multimers, and particularly as hetero-dimers or tetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four-chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer ofbasic four-chain units held together by disulfide bonds. IgA globulin,and occasionally IgG globulin, may also exist in multimeric form inserum. In the case of multimer, each four-chain unit may be the same ordifferent.

Various exemplary assembled ligand-immunoglobulin chimeras within thescope herein are schematically diagrammed below:

(a) AC_(L) -AC_(L) ;

(b) AC_(H) -(AC_(H), AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H));

(c) AC_(L) -AC_(H) -(AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), V_(L) C_(L)-AC_(H), or V_(L) C_(L) -V_(H) C_(H));

(d) AC_(L) -V_(H) C_(H) -(AC_(H), or AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H));

(e) V_(L) C_(L) -AC_(H) -(AC_(L) -V_(H) C_(H), or V_(L) C_(L) -AC_(H));and

(f) (A-Y_(n) -(V_(L) C_(L) -V_(H) C_(H))₂,

wherein

each A represents identical or different amino acid sequences capable ofselective binding to said receptor;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the ligand sequences of the present invention can beinserted between immunoglobulin heavy chain and light chain sequencessuch that an immunoglobulin comprising a chimeric heavy chain isobtained. In this embodiment, the ligand sequences are fused to the 3'end of an immunoglobulin heavy chain in each arm of an immunoglobulin,either between the hinge and the CH2 domain, or between the CM2 and CH3domains. Similar constructs have been reported by Hoogenboom, H. R. etal., Mol. Immunol. 28, 1027-1037 (1991).

The ligand-immunoglobulin chimeras of the present invention areconstructed in a fashion similar to the construction of bispecificantibodies, such as, for example, disclosed in EP 125,023 (published 14Nov. 1984); U.S. Pat. No. 4,444,878 (issued 24 Apr. 1984); (Munro, A,,Nature 312, 597 (1984); Morrison, et al., Science 229, 1202-1207 (1985);Berg et al., Proc. Natl. Acad. Sci. USA 88, 4723-4727 (1991).

The DNA encoding a native ligand herein may be obtained from any cDNAlibrary prepared from tissue believed to possess mRNA for the desiredligand and to express it at a detectable level. Libraries are screenedwith probes designed to identify the gene of interest or the proteinencoded by it. For cDNA expression libraries, suitable probes usuallyinclude mono- and polyclonal antibodies that recognize and specificallybind to the desired protein; oligonucleotides of about 20-80 bases inlength that encode known or suspected portions of the ligand cDNA fromthe same or different species; and/or complementary or homologous cDNAsor their fragments that encode the same or similar gene.

An alternative means to isolate the gene encoding a desired nativeligand is to use polymerase chain reaction (PCR) methodology asdescribed in U.S. Pat. No. 4,683,195, issued 28 Jul. 1987, in section 14of Sambrook et al., Molecular Cloning: A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory Press. New York, 1989, or inChapter 15 of Current Protocols in Molecular Biology, Ausubel et al.eds., Greene Publishing Associates and Wiley-Interscience 1991.

Another alternative is to chemically synthesize the gene encoding thedesired (native or variant) ligand using one of the methods described inEngels et al., Agnew. Chem. Int. Ed. Engl. 28, 716 (1989). These methodsinclude triester, phosphite, phosphoramidite and H-Phosphonate methods,PCR and other autoprimer methods, and oligonucleotide syntheses on solidsupports. These methods maybe used if the entire nucleic acid sequenceof the gene is known, or the sequence of the nucleic acid complementaryto the coding strand is available, or, alternatively, if the targetamino acid sequence is known, one may infer potential nucleic acidsequences, using known and preferred coding residues for each amino acidresidue.

The amino acid sequence variants of the ligands of this invention arepreferably constructed by mutating the DNA sequence that encodes theprotein core of a wild-type ligand. Generally, particular regions orsites of the DNA, identified by methods discussed hereinabove, will betargeted for mutagenesis, and thus the general methodology employed toaccomplish this is termed site-directed mutagenesis. The mutations aremade using DNA modifying enzymes such as restriction endonucleases(which cleave DNA at particular locations), nucleases (which degradeDNA) and/or polymerases (which synthesize DNA).

The following is a brief discussion of certain commonly used techniquesof recombinant DNA technology that can be used for making the liganddimers of the present invention. These and similar techniques areequally suitable for making variants of receptor binding domains ofnative ligands, fusions of native and variant ligands, with or without alinker, including linkers of immunoglobulin origin. Further details ofthese and similar techniques are found in general textbooks, such as,for example, Sambrook et al., supra, and Current Protocols in MolecularBiology, Ausubel et al. eds., supra.

Site Directed Mutagenesis

Preparation of ligand variants and of dimers including such ligandvariants in accordance herewith is preferably achieved by site-specificmutagenesis of DNA that encodes an earlier prepared variant or anonvariant version of the protein. Site-specific mutagenesis allows theproduction of variants through the use of specific oligonucleotidesequences that encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the junction being traversed. Typically, aprimer of about 20 to 25 nucleotides in length is preferred, with about5 to 10 residues on both sides of the junction of the sequence beingaltered. In general, the technique of site-specific mutagenesis is wellknown in the art as exemplified by publications such as Adelman et al.,DNA, 2: 183 (1983).

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981). Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Alternatively, plasmid vectors thatcontain a single-stranded phage origin of replication (Veira et al.,Meth. Enzymol., 153: 3 (1987)) may be employed to obtain single-strandedDNA.

In general, site-directed mutagenesis may, for example, be performed byfirst obtaining a single-stranded vector that includes within itssequence a DNA sequence that encodes the relevant selectin. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA), 75: 5765 (1978). This primer is thenannealed with the single-stranded ligand sequence-containing vector, andsubjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells and clones are selected, via hybridization toa radioactive probe consisting of the ³² P-labeled mutagenesis primer,that include recombinant vectors bearing the mutated sequencearrangement.

After such a clone is selected, the mutated region may be removed andplaced in an appropriate vector for the production of the desiredvariant, generally an expression vector of the type that typically isemployed for transformation of an appropriate eukaryotic host. In thecontext of the present invention, Chinese hamster ovary (CHO) cells or293 (human kidney cells described by Graham et al., J. Gen. Virol., 36:59 (1977)) are preferred for the preparation of long-term stablepolypeptide producers. However, the invention is not limited to CHOproduction, as it is known that numerous other cell types are suitablyemployed, particularly where one desires only transient production ofthe enzyme for test purposes. For example, described below is atransient system employing 293 cells that provides a convenient systemfor production of ligand variants or ligand dimers, e.g.ligand-immunoglobulin chimeras for analytical purposes.

Another method for making mutations in the DNA sequence encoding aligand involves cleaving the DNA at the appropriate position bydigestion with restriction enzymes, recovering the properly cleaved DNA,synthesizing an oligonucleotide encoding the desired amino acid andflanking regions such as polylinkers with blunt ends (or, instead ofusing polylinkers, digesting the synthetic oligonucleotide with therestriction enzymes also used to cleave the ligand-encoding DNA, therebycreating cohesive termini), and ligating the synthetic DNA into theremainder of the ligand-encoding structural gene.

PCR Mutagenesis

PCR mutagenesis is also suitable for making ligands, including liganddimers for practicing the present invention. While the followingdiscussion refers to DNA, it is understood that the technique also findapplication with RNA. The PCR technique generally refers to thefollowing procedure. When small amounts of template DNA are used asstarting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers is designed to overlap the position of the mutationand to contain the mutation; the sequence of the other primer must beidentical to a stretch of sequence of the opposite strand of theplasmid, but this sequence can be located anywhere along the plasmidDNA. It is preferred, however, that the sequence of the second primer islocated within 200 nucleotides from that of the first, such that in theend the entire amplified region of DNA bounded by the primers can beeasily sequenced. PCR amplification using a primer pair like the onejust described results in a population of DNA fragments that differ atthe position of the mutation specified by the primer, and possibly atother positions, as template copying is somewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more) partligation.

Host Cell Cultures and Vectors

Although expression on Chinese hamster ovary (CHO) cells and in thehuman embryonic kidney cell line 293 (Urlaub and Chasin, Proc. Natl.Acad. Sci. USA 77, 4216 (1980); Graham et al., J. Gen. Virol., 36, 59(1977)) are ultimately preferred, the vectors and methods disclosedherein are suitable for use in host cells over a wide range ofeukaryotic organisms.

In general, prokaryotes are preferred for the initial cloning of DNAsequences and constructing the vectors useful in the invention. Forexample, E. coli K12 strain 294 (ATCC No. 31,446) and E. coli strainW3110 (ATCC No. 27,325) are particularly useful. Other suitablemicrobial strains include E. coli strains such as E. coli B, and E. coliX1776 (ATCC No. 31,537). These examples are, of course, intended to beillustrative rather than limiting.

Prokaryotes also are useful for expression. The aforementioned strains,as well as bacilli such as Bacillus subtilis, and otherenterobacteriaceae such as, e.g., Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species are examples of useful hostsfor expression.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., Gene, 2: 95 (1977)). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, must also contain, or be modified tocontain, promoters that can be used by the microbial organism forexpression of its own proteins.

Those promoters most commonly used in recombinant DNA constructioninclude β-lactamase (penicillinase) and lactose promoter systems (Changet al., Nature, 375: 615 (1978); Itakura et al., Science, 198: 1056(1977); Goeddel et al., Nature, 281: 544 (1979)) and a tryptophan (trp)promoter system (Goeddel et al., Nucl. Acids Res., 8: 4057 (1980); EPOAppl. Publ. No. 36,776), and the alkaline phosphatase systems. Whilethese are the most commonly used, other microbial promoters have beendiscovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors (see, e.g., Siebenlist et al., Cell,20: 269 (1980)).

In addition to prokaryotes, eukaryotic microbes, such as yeasts, alsoare suitably used herein. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among eukaryotic microorganisms,although a number of other strains are commonly available. For example,for expression in Saccharomyces, the plasmid YRp7 (Stinchcomb et al.,Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemperet al., Gene, 10: 157 (1980)) is commonly used. This plasmid alreadycontains the trp1 gene that provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44,076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presenceof the trp1 lesion as a characteristic of the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073(1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.,7: 149 (1968); Holland et al., Biochemistry, 17: 4900 (1978)), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In the construction ofsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector 3' of thesequence desired to be expressed to provide polyadenylation of the mRNAand termination. Other promoters that have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)). Examples of such useful host cell linesare VERO and HeLa cells, CHO cell lines, and W138, BHK, COS-7, (ATCC CRL1651), 293, and MDCK (ATCC CCL 34) cell lines. Expression vectors forsuch cells ordinarily include (if necessary) an origin of replication, apromoter located in front of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment that also contains the SV40 viral origin of replication (Fierset al., Nature, 273: 113 (1978)). Smaller or larger SV40 fragments arealso suitably used, provided there is included the approximately 250-bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication typically is provided either by construction ofthe vector to include an exogenous origin, such as may be derived fromSV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or by thehost cell chromosomal replication mechanism. If the vector is integratedinto the host cell chromosome, the latter is often sufficient.

Satisfactory amounts of ligand variants or dimers are produced by cellcultures; however, refinements, using a secondary coding sequence, serveto enhance production levels even further. The secondary coding sequencecomprises dihydrofolate reductase (DHFR) that is affected by anexternally controlled parameter, such as methotrexate (MTX), thuspermitting control of expression by control of the MTX concentration.

In the selection of a preferred host cell for transfection by thevectors of the invention that comprise DNA sequences encoding bothvariant selectin and DHFR protein, it is appropriate to consider thetype of DHFR protein employed. If wild-type DHFR protein is employed, itis preferable to select a host cell that is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is the CHOcell line deficient in DHFR activity, prepared and propagated, asdescribed by Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77: 4216(1980).

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant to MTX,MTX-containing media can be used as a means of selection, provided thatthe host cells are themselves MTX sensitive. Most eukaryotic cells thatare capable of absorbing MTX appear to be sensitive to MTX. One suchuseful cell line is a CHO line, CMO-K1 (ATCC No. CCL 61).

Typical Cloning and Expression Methodologies Available

If mammalian cells are used as host cells, transfection generally iscarried out by the calcium phosphate precipitation method as describedby Graham and Van der Eb, Virology, 52: 546 (1978). However, othermethods for introducing DNA into cells such as nuclear injection,electroporation, or protoplast fusion are also suitably used.

If yeast are used as the host, transfection is generally accomplishedusing polyethylene glycol, as taught by Hinnen, Proc. Natl. Acad. Sci.U.S.A., 75: 1929-1933 (1978).

If prokaryotic cells or cells that contain substantial cell wallconstructions are used, the preferred method of transfection is calciumtreatment using calcium as described by Cohen et al., Proc. Natl. Acad.Sci. (USA) 69: 2110 (1972), or more recently electroporation.

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to form the plasmids required.

Cleavage is performed by treating with restriction enzyme (or enzymes)in suitable buffer. In general, about 1 μg plasmid or DNA fragments isused with about 1 unit of enzyme in about 20 μl of buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer.) Incubation times of about one hour at 37° C. areworkable. After incubation, protein is removed by extraction with phenoland chloroform, and the nucleic acid is recovered from the aqueousfraction by precipitation with ethanol.

If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of the Klenow fragment of DNA PolymeraseI (Klenow), phenol-chloroform extracted, and ethanol precipitated.

Size separation of the cleaved fragments is performed using 6 percentpolyacrylamide gel described by Goeddel et al., Nucleic Acids Res., 8:4057 (1980).

For ligation, approximately equimolar amounts of the desired components,suitably end tailored to provide correct matching, are treated withabout 10 units T4 DNA ligase per 0.5 μg DNA. (When cleaved vectors areused as components, it may be useful to prevent religation of thecleaved vector by pretreatment with bacterial alkaline phosphatase.)

As discussed above, ligand variants are preferably produced by means ofspecific mutation. Variants useful in the practice of the presentinvention are formed most readily through the use of specificoligonucleotide sequences that encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a sequence of sufficient size and sequence complexity to form astable duplex on both sides of the mutation being traversed.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are typically used to transform E. coli K12 (ATCC31,446) or other suitable E. coli strains, and successful transformantsselected by ampicillin or tetracycline resistance where appropriate.Plasmids from the transformants are prepared and analyzed by restrictionmapping and/or DNA sequencing by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods ofEnzymology, 65: 499 (1980).

After introduction of the DNA into the mammalian cell host and selectionin medium for stable transformants, amplification of DHFR-protein-codingsequences is effected by growing host cell cultures in the presence ofapproximately 20,000-500,000 nM concentrations of MTX, a competitiveinhibitor of DHFR activity. The effective range of concentration ishighly dependent, of course, upon the nature of the DHFR gene andprotein and the characteristics of the host. Clearly, generally definedupper and lower limits cannot be ascertained. Suitable concentrations ofother folic acid analogs or other compounds that inhibit DHFR could alsobe used. MTX itself is, however, convenient, readily available, andeffective.

In a particular embodiment, ligand-immunoglobulin chimeric molecules areused in accordance with the present invention. The ligand-immunoglobulinchimeras preferably are recovered from the culture medium as a secretedprotein, although it also may be recovered from host cell lysates whendirectly expressed without a secretory signal. When the chimera isexpressed in a recombinant cell other than one of human origin, thevariant is thus completely free of proteins of human origin. However, itis necessary to purify the variant from recombinant cell proteins inorder to obtain preparations that are substantially homogeneous as toprotein. As a first step, the culture medium or lysate is centrifuged toremove particulate cell debris.

The chimera is then purified from contaminant soluble proteins, forexample, by an appropriate combination of conventional chromatographymethods, e.g. gel filtration, ion-exchange, hydrophobic interaction,affinity, immunoaffinity chromatography, reverse phase HPLC;precipitation, e.g. ethanol precipitation, ammonium sulfateprecipitation, or, preferably, immunoprecipitation with anti-HGF(polyclonal or monoclonal) antibodies covalently linked to Sepharose.Due to its high affinity to heparin, HGF can be conveniently purified ona heparin, such as heparin-Sepharose column. One skilled in the art willappreciate that purification methods suitable for native HGF may requiremodification to account for changes in the character of HGF or itsvariants upon expression in recombinant cell culture.

In a further embodiment, the two (identical or different) ligands arelinked with a non-immunoglobulin linker. The linker may be residue of acovalent cross-linking agent capable of linking the ligands without theimpairment of the receptor binding function or a linkage the formationof which is induced by such cross-linking agents. A concise review ofcovalent cross-linking reagents, including a guide to the selection ofsuch reagents and methods for their preparation are provided by Tae, H.Jr. in Meth. Enzymol. 580-609 (1983) and in the references citedtherein. The selection of the most appropriate reagent for a specificpurpose from the wide variety of cross-linking agents available, is wellwithin the skill of an ordinary artisan.

In general, zero-length, homo- or heterobifunctional cross-linkingagents are preferred for the purpose of the present invention.Zero-length cross linking reagents induce the direct conjugation of twoligands without the introduction of any extrinsic material. Agents thatcatalyze the formation of disulfide bonds belong in this category.Another example is reagents that induce the condensation of carboxy andprimary amino groups to form an amide bond, such as carbodiimides,ethylchloroformate, Woodward's reagent K1, carbonyldiimidazole, etc.Homobifunctional reagents carry two identical functional groups, whereasheterobifunctional reagents contain two dissimilar functional groups. Avast majority of the heterobifunctional cross-linking agents contains aprimary amine-reactive group and a thiol-reactive group. A novelheterobifunctional linker for formyl to thiol coupling was disclosed byHeindel, N. D. et al., Bioconjugate Chem. 2, 427-430 (1991). In apreferred embodiment, the covalent cross-linking agents are selectedfrom reagents capable of forming disulfide (--S--S--), glycol(--CH(OH)--CH(OH)--), azo (--N═N--), sulfone (--S(═O₂)--), or ester(--C(═O)--O--) bridges.

In a different approach, the ligands are linked via theiroligosaccharides. Chemical or enzymatic oxidation of oligosaccharides onpolypeptide ligands to aldehydes yields unique functional groups on themolecule which can react with compounds containing, for example, amineshydrazines, hydrazides, or semicarbazides. Since the glycosylationssites are well defined in polypeptide molecules, selective coupling viaoxidized oligosaccharide moieties will yield in a more uniform productthan other coupling methods, and is expected to have less adverse effecton the receptor binding properties of the ligands. Carbohydrate-directedheterobifunctional cross-linking agents are, for example, disclosed incopending patent application Ser. No. 07/926,077 filed 5 Aug. 1992.

It will be understood that the coupling of more than two ligandsequences with various linked sequences, e.g., cross-linking reagents ispossible, and is within the scope of the present invention.

In a further embodiment, two or more ligands are connected bypolypeptide linker sequences, and accordingly, are presented to theirreceptor as a single-chain multifunctional polypeptide molecule. Thepolypeptide linker functions as a "spacer" whose function is to separatethe functional ligand domains so that they can independently assumetheir proper tertiary conformation. The polypeptide linker usuallycomprises between about 5 and about 25 residues, and preferably containsat least about 10, more preferably at least about 15 amino acids, and iscomposed of amino acid residues which together provide a hydrophilic,relatively unstructured region. Linking amino acid sequences with littleor no secondary structure work well. If desired, one or more uniquecleavage sites recognizable by a specific cleavage agent (e.g. protease)may be included in the polypeptide linker. The specific amino acids inthe spacer can vary, however, cysteines should be avoided. The spacersequence may mimic the tertiary structure of an amino acid sequencenormally linking two receptor binding domains in a native bifunctionalligand. It can also be designed to assume a desired structure, such as ahelical structure. Suitable polypeptide linkers are, for example,disclosed in WO 88/09344 (published 1 Dec. 1988), as are methods for theproduction of multifunctional proteins comprising such linkers.

In a further specific embodiment, the ligands are dimerized byamphiphilic helices. It is known that recurring copies of the amino acidleucine (Leu) in gene regulatory proteins can serve as teeth that "zip"two protein molecules together to provide a dimer. Leucine zipper wasfirst discovered when a small segment of the protein C/EBP was fit intoa hypothetical alpha helix. Surprisingly, the leucines, which make upevery seventh amino acid in this protein, lined up in a column.Subsequently, two additional, C/EBP related proteins were identified andshown to have a similar function. One of them, GCN4 is a gene regulatoryprotein from yeast, the other one, is the product of a proto-oncogenejun. It has been found that zipper regions associate in parallel whenthey combine, i.e. the leucines on apposed molecules line up side byside. It has also been shown that non-identical proteins my be zipperedto provide heterodimers. Such leucine zippers are particularly suitablefor preparing ligand dimers within the scope of the invention.Alternatively, the sequence of the amphipathic helix may be taken from afour-helix bundle design, essentially as described by Pack P. andPluckthun, A., Biochemistry 31, 1579-1584 (1992). For further detailsabout molecular, e.g. leucine zippers, which can serve as heterologouslinkers for the purpose of the present invention, see for example:Landshculz, W. H., et al. Science 240, 1759-1764 (1988); O'Shea, E. K.et al., Science 243, 538-542 (1989); McKnight, S. L., ScientificAmerican 54-64, April 1991; Schmidt-Dorr. T. et al., Biochemistry 30,9657-9664 (1991); Blondel, A. and Bedouelle, H. Protein Engineering 4,457-461 (1991), Pack, P. and Pluckthun, A., supra, and the referencescited in these papers.

In a specific embodiment, the present invention provides methods for theconversion of ligand variants capable of binding their receptors buthaving no or diminished receptor activating ability to potent agonist ofthe respective native ligands. The target ligands preferably retainsubstantially full receptor binding affinity of the native ligand.

The expression "retain substantially full receptor binding affinity ofnative ligand" and grammatical variant thereof as used herein mean thatthe receptor binding affinity of the ligand variant is not less thenabout 70%, preferably not less than about 80%, more preferably not lessthan about 90%, most preferably not less than about 95% of the affinitywith which the corresponding native ligand binds its receptor.

Receptor binding can be determined in standard assays, such as, forexample, in the competitive binding assay disclosed in the examples.

The terms "substantially incapable of receptor activation", and"substantially devoid of biological activity" mean that the activityexhibited by a variant ligand is less than about 20%, preferably lessthan about 15%, more preferably less than about 10%, most preferablyless than about 5% of the respective activity of the correspondingnative ligand in an established assay of receptor activation or ligandbiological activity.

The operability of the present invention was first demonstrated byactivating the receptor for hepatocyte growth factor (HGFr) withchimeric molecules formed by the fusion of wild-type HGF ligands andtheir amino acid sequence variants to immunoglobulin constant domainsequences.

The HGF biological activity may, for example, be determined in an invitro or in vivo assay of hepatocyte growth promotion. Adult rathepatocytes in primary culture have been extensively used to search forfactors that regulate hepatocyte proliferation. Accordingly, themitogenic effect of an HGF variant can be conveniently determined in anassay suitable for testing the ability of an HGF molecule to induce DNAsynthesis of rat hepatocytes in primary cultures, such as, for example,described in Example 2. Human hepatocytes are also available from wholeliver perfusion of organs deemed unacceptable for transplantation,pare-downs of adult livers used for transplantation in children, fetallivers and liver remnants removed at surgery for other indications.Human hepatocytes can be cultured similarly to the methods establishedfor preparing primary cultures of normal rat hepatocytes. Hepatocyte DNAsynthesis can, for example, be assayed by measuring incorporation of (³H)thymidine into DNA, with appropriate hydroxyurea controls forreplicative synthesis.

The effect of HGF variants on hepatocyte growth can also be tested invivo in animal models of liver dysfunction and regeneration, such as inrats following partial hepatectomy, or carbon tetrachloride causedhepatic injury, in D-galactosamine induced acute liver failure models,etc. According to a suitable protocol, a liver poison, e.g.α-naphthylisothiocyanate (ANIT) is administered to rats in apredetermined concentration capable of causing reproducible significantelevation of liver enzyme and bilirubin levels. The rats are thentreated with the HGF variant to be tested, sacrificed and the liverenzyme and bilirubin levels are determined. The livers are additionallyobserved for hepatic lesions.

The biological activity of other ligands and ligand variants can beassayed by methods known in the art.

The compounds of the present invention are able to activate theirrespective receptors and thereby mimic the biological activity of thecorresponding native ligands. They can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thelinked ligand variants are combined in admixture with a pharmaceuticallyacceptable carrier. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed., 1980, MackPublishing Co., edited by Oslo et al. These compositions will typicallycontain an effective amount of the compound, for example, from on theorder of about 0.5 to about 10 mg/ml, together with a suitable amount ofcarrier to prepare pharmaceutically acceptable compositions suitable foreffective administration to the patient. The compounds may beadministered parenterally or by other methods that ensure its deliveryto the bloodstream in an effective form, essentially following routes ofadministration known for the corresponding native ligands.

Compositions particularly well suited for the clinical administration ofthe compounds o the present invention are the same as or can bedeveloped based upon formulations known for the corresponding nativeligands.

Dosages and desired drug concentrations of pharmaceutical compositionsmay vary depending on the particular use envisioned. Preliminary dosagescan be determined in animal tests, and interspecies scaling of dosagescan be performed in a manner known in the art, e.g. as disclosed inMordenti et al., Pharmaceut. Res. 8, 1351 (1991) and in the referencescited therein.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

EXAMPLE 1 Recombinant Production of the huHGF Variants

A. Site-directed mutagenesis

Plasmid DNA isolation, polyacrylamide and agarose gel electrophoresiswere performed as disclosed in Sambrook et al., supra.

Mammalian expression plasmid pRK 5.1 with a CMV promotor (Genentech,Inc.) was used for mutagenesis of huHGF allowing secretion of the HGFvariants in the culture medium and directly assayed for biologicalactivity and binding. This expression vector is a derivative of pRK5,the construction of which is disclosed in EP 307,247 published 15 Mar.1989. pRK 5.1 was derived from RK5 by insertion of theself-complementary oligonucleotide 5'-AGCTTGCCTCGAGGCA-3' (SEQ. ID. NO:14). The nucleotide sequence encoding this the pRK 5.1 vector isdisclosed in copending application Ser. No. 07/885,971 filed 18 May 1992now U.S. Pat. No. 5,328,837.

The huHGF cDNA used corresponds to the 728 amino acid form as publishedearlier (Miyazawa et al., 1989, supra).

Mutagenesis was performed according to the method of Kunkel using thecommercially available dut- ung- strain of E. coli (Kunkel et al.,Method. Enzymol. 154, 367-382 (1987)). Synthetic oligonucleotides usedfor in vitro mutagenesis and sequencing primers were prepared using theApplied Biosystem 380A DNA synthesizer as described (Matteucci et al.,J. Am. Chem. Soc. 103, 3185-3191 (1981)). For generation of the desiredmutants, oligonucleotides of sequences coding for the desired amino acidsubstitutions were synthesized and used as primers. The oligonucleotideswere annealed to single-stranded pRK 5.1-huHSA that had been prepared bystandard procedures (Viera et al., Method. Enzymol. 142, 3 (1987)).

A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP),deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), was combinedwith a modified thio-deoxyribonuleosine called dCTP(aS) provided in thekit by the manufacturer, and added to the single stranded pRK 5.1-huHGFto which was annealed the oligonucleotide.

Upon addition of DNA polymerase to this mixture, a strand of DNAidentical to pRK 5.1-huHGF except for the mutated bases was generated.In addition, this new strand of DNA contained dCTP(aS) instead of dCTP,which served to protect from restriction endonuclease digestion. Afterthe template strand of the double-stranded heteroduplex was nicked withan appropriate restriction enzyme, the template strand was digested withExoIII nuclease past the region that contained the mutagenic oligomer.The reaction was then stopped to leave a molecule hat was only partlysingle-stranded. A complete double-stranded DNA homoduplex molecule wasthen formed by DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase.

The following oligonucleotides were prepared to use as primers togenerate pRK 5.1-huHGF variant molecules:

    __________________________________________________________________________    R494E huHGF:                                                                          TTGGAATCCCATTTACAACCTCGAGTTGTTTCGTTTTGGCACAAGAT                                                                (SEQ. ID. NO: 1)                     R494D huHGF:                                                                          GAATCCCATTTACGACGTCCAATTGTTTCG   (SEQ. ID. NO: 2)                     R494A huHGF:                                                                          CCCATTTACAACTGCCAATTGTTTCG       (SEQ. ID. NO: 3)                     Q534H huHGF:                                                                          AGAAGGGAAACAGTGTCGTGCA           (SEQ. ID. NO: 4)                     Y673S huHGF:                                                                          AGTGGGCCACCAGAATCCCCCT           (SEQ. ID. NO: 5)                     V692S huHGF:                                                                          TCCACGACCAGGAGAAATGACAC          (SEQ. ID. NO: 6)                     ΔK1 huHGF:                                                                      GCATTCAACTTCTGAGTTTCTAATGTAGTC   (SEQ. ID. NO: 7)                     ΔK2 huHGF:                                                                      CATAGTATTGTCAGCTTCAACTTCTGAACA   (SEQ. ID. NO: 8)                     ΔK3 huHGF:                                                                      TCCATGTGACATATCTTCAGTTGTTTCCAA   (SEQ. ID. NO: 9)                     ΔK4 huHGF:                                                                      TGTGGTATCACCTTCATCTTGTCCATGTGA   (SEQ. ID. NO: 10)                    N-303 huHGF:                                                                          ACCTTGGATGCATTAAGTTGTTTC         (SEQ. ID. NO: 11)                    N-384 huHGF:                                                                          TTGTCCATGTGATTAATCACAGT          (SEQ. ID. NO: 12)                    α-chain:                                                                        GTTCGTGTTGGGATCCCATTTACCTATCGCAATTG                                                                            (SEQ. ID. NO: 13)                    __________________________________________________________________________

The Y673S, V692S huHGF variant was obtained from wild-type huHGF as atemplate, using both oligonucleotides used for generating the twomutations.

The mutant huHGF constructs generated using the protocol above weretransformed in E. coli host strain MM294tonA using the standard calciumchloride procedure (Sambrook et al., supra) for preparation andtransformation of competent cells. MM294tonA (which is resistant to T1phage) was prepared by the insertion and subsequent imprecise excisionof a Tn10 transposon into the tonA gene. This gene was then inserted,using transposon insertion mutagenesis (Kleckner et al., J. Mol. Biol.116, 125-159 (1977)), into E. coli host MM294 (ATCC 31,446).

The DNA extract from individual colonies of bacterial transformantsusing the standard miniprep procedure of Sambrook et al., supra. Theplasmids were further purified by passage over a Sephacryl CL6B spincolumn, and then analyzed by sequencing and by restriction endonucleasedigestion and agarose gel electrophoresis.

B. Transfection of Human Embryonic Kidney 293 Cells

Plasmids with the correct sequence were used to transfect human fetalkidney 293 cells by the calcium phosphate method. 293 cells were growthto 70% confluence in 6-well plates. 2.5 μg of huHGF plasmid DNA variantwas dissolved in 150 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227M CaCl₂.Added to this (dropwise while vortexing) was 150 μl of 50 mM HEPESbuffer (pH 7.35), 280 mM NaCl, 1.5 mM NAPO₄, and the precipitate wasallowed to form for ten minutes at 25° C. The suspended precipitate wasthem added to the cells in the individual wells in a 6-well plate. Thecell monolayers were incubated for 4 hours in the presence of the DNAprecipitate, washed once with PBS, and cultured in serum-free medium for72 h. When stable populations were made, the HGF cDNA was subcloned inan episomal CMV driven expression plasmid pCisEBON (G. Cachianes, C, Ho,R. Weber, S. Williams, D. Goeddel, and D. Lueng, in preparation).pCisEBON is a pRK5 derivative that includes sequences encoding aselectable marker gene encoding neomycin phosphotransferase (NEO), anorigin of replication derived from Epstein Bart virus origin (ori P) andthe viral EBNA-1 gene. The product of the EBNA-1 gene promotes stable,episomal replication of plasmids containing the ori P sequence seeCachianes, G. et al., Technique, in press (1992)!. The nucleotidesequence encoding pCisEBON is disclosed in copending application Ser.No. 07/885,971 filed 18 May 1992. The populations were directly selectedin Neomycin selective medium.

EXAMPLE 2 Assay Methods

In view of the pleiotropic activities of HGF, a molecule with astructure unlike any other known growth factor, it is important tounderstand the molecular interaction of this factor with its receptor.The huHGF variants produced as described in Example 1 were analyzed fortheir ability to induce DNA synthesis of hepatocytes in primary cultureand to compete for binding to a soluble form of the huHGF receptor.

A. Protein quantification of wild-type huHGF and huHGF variants

A specific two-site huHGF sandwich ELISA using two monoclonal antibodieswas used to quantify wild-type recombinant huHGF (WT rhuHGF), singlechain and protease substitution variants. Microtiter plates (Maxisorb,Nunc) were coated with 10 mg/ml of a monoclonal anti-rhuHGF antibody A3.1.2 (IgG2a phenotype, affinity: 3.2×10⁻⁸ mol) in 50 mM Carbonatebuffer, pH 9.6, overnight at 4° C. After blocking plates with 0.5% BSA(Sigma), 0.01% thimerosal in PBS, pH 7.4, and subsequent washes,duplicate serial dilutions of HGF samples were prepared and in parallela CHO-expressed rhuHGF (40-0.1 ng/mL) was used as a standard. Fiftymicroliters of these dilutions were simultaneously incubated with 50 mLof a 1:1500 diluted horseradish peroxidase conjugated monoclonalanti-rhuHGF antibody B 4.3 (IgG1 phenotype, affinity: 1.3×10⁻⁸ mol) for2 h at RT. The substrate was prepared by adding 0.04%o-phenylenediamine-dihydrochloride (Sigma) and 0.012% (v/v)hydrogen-peroxide (Sigma) to PBS and 100 ml were added to the washedplates for 15 minutes at RT. The reaction was stopped by adding 50 mL of2.25M sulfuric acid to each well. The absorbance at 490 nm, with theabsorbance at 405 nm subtracted as background, was determined on amicrotiter plate reader (Vmax, Molecular Devices, Menlo Park, Calif.).The data was reduced using a four-parameter curve-fitting programdeveloped at Genentech, Inc.

An HGF polyclonal sandwich ELISA was used to quantify all kringledeletion and C-terminal truncation variants. Briefly, microtiter plates(Nunc) were coated with 5 mg/mL guinea pig polyclonal (antiCHO-expressed rhuHGF) IgG antibody preparation (Genentech, Inc.) asdescribed above. This antibody recognizes rhuHGF as well as HGFtruncated forms when compared to visual inspection of Western blots,making it ideal for monitoring HGF variants. Plates were blocked andduplicate serial dilutions of 293 cell supernatants (1:103-6.106) wereadded and incubated over night at 4° C. Purified CHO-expressed rhuHGF(100-0.78 ng/mL) was used as a standard and incubated in parallel.Plates were washed and incubated with a 1:500 dilution of the samepolyclonal antibody (approx. 400 ng/mL) but in this case horseradishperoxidase conjugated for detection of the variants (see above). Westernblotting was performed to determine the size of the expressed HGFvariants. For this, SDS-polyacrylamide gel electrophoresis and Westernblotting were performed using standard methods with the polyclonal IgGantibody preparation (500 ng/mL). A chemiluminescent detection method(Amersham) and a goat anti-guinea pig IgG-horseradish peroxidaseconjugate (1:5000) were used for development of the blot as described bythe manufacturer.

B. Soluble HGF receptor binding assay

Previous studies on HGF binding to hepatocytes have shown that havehuHGF could bind to its cell surface receptor with high affinity(Kd-24-32 pM; Higuchi and Nakamura, Biochem. Biophys. Res. Comm. 174,831-838 (1991)). We preferred to examine HGF binding using a solubleform of the receptor because of the nonspecific binding of HGF to cellsurface heparin sulfate proteoglycans (Naldini et al., EMBO J. 10,2867-2878 (1991)).

Cell supernatants (concentrated on Amicon filters if concentration wasbelow 600 ng/mL) were tested for their ability to block in solution thebinding of CHO-expressed ¹²⁵ I rhuHGF (2-5×103 Ci/mmole, kindly providedby T. Zioncheck, Genentech, Inc.) to the extracellular domain of thehuman HGF receptor (huHGFr) fused to the Fc constant region of an humanIgG, expressed and secreted from 293 cells.

1. Construction of huHGFr-IgG chimeras

A full length cDNA clone encoding the huHGFr was constructed by joiningpartial cDNAs isolated from cDNA libraries and from PCR amplification.Coding sequences for amino acids 1-270 were isolated from a humanplacental cDNA library (provided by T. Mason, Genentech) screened with a50 mer oligonucleotide(5'-ATGAAGGCCCCCGCTGTGCTTGCACCTGGCATCCTCGTGCTCCTGTTTACC-3') (SEQ. ID.NO: 15). Sequences encoding amino acids 809-1390 were isolated from ahuman liver library (Stragagen) screened with the oligonucleotide probe(5'-CACTAGTTAGGATGGGCTTACATGTCTGTCAGAGGATACTTCACTTGTCGGCATGAA CCGT-3').(SEQ. ID. NO: 16)

Conditions for plating libraries, and for hybridization and washingfilters were as described (Godowski et al., Proc. Natl. Acad. Sci. USA86, 8083-8087 (1989)). PCR was used to isolate a cDNA clone containingresidues 271-808 of the HGFr (c-met) from A549 cells. Ten μgs of totalRNA was used for reverse transcription using a primer specific to theHGFr (5'-TAGTACTAGCACTATTATTTCT -3') (SEQ. ID. NO: 17) in a 100 μlreaction using Moloney murine leukemia virus reverse transcriptase andbuffers supplied by Bethesda Research Laboratories. One-tenth of thisreaction mixture was used for PCR amplification. The PCR reaction wasperformed in a volume of 100 μl containing 10 μl of the reversetranscriptase reaction, 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM(NH4)SO4, 6 mM MgSO4, 0.1% Trition X-100, 1 U of Vent DNA polymerase(New England Biolabs) and 50 pmol each of the forward primer(5'-TTACTTCTTGACGGTCCAAG-3' (SEQ. ID. NO: 18) and the reverse primer(5'-CAGGGGAGTTGCAGATTCAGCTGT-3') (SEQ. ID. NO: 19). After thirty cyclesof denaturation (95° C., 1 min), annealing (55° C., 45 secs) andextension (72° C., 2 min), the PCR product were recovered fromlow-melting temperature agarose gels. The full-length HGFr cDNA wassubcloned into vector pRK7 (see WO 90/02798, published 22 Mar. 1990) anddouble-stranded DNA sequencing was performed by the dideoxynucleotidemethod.

The coding sequence of the extracellular domain of the huHGFr was fusedto those of the human IgG1 heavy chain in a two-step process. PCR wasused to generate a fragment with a unique BstEII site 3' to the codingsequences of the HGFr amino acid 929. The 5' primer (located in thevector upstream of the HGFr coding sequences) and the 3' primer(5'-AGTTTTTGTCGGTTGACCTGATCATTCTGATCTGGTTGAACTATTAC-3') (SEQ. ID. NO:20) were used in a 100 μl reaction as described above except that theextension time at 72° C. was 3 minutes, and 40 ng of the full lengthHGFr expression vector was used as template. Following amplification,the PCR product was joined to the human IgG-γ1 heavy chain cDNA througha unique BstEII site in that construct (Bennett et al., J. Biol. Chem.266, 23060-23067 (1991)). The resulting construct contained the codingsequences of amino acids 1-929 of the huHGFr fused via the BstEII site(adding the coding sequences for amino acids V and T) to the codingsequences of amino acids 216-443 of the human IgG-γ1 heavy chain.Sequencing of the construct was carried out as described above.

2. Binding assay

The binding assay was performed in breakable microtiter plates (Nunc)coated o/n at 4° C. with 1 mg/mL of rabbit-anti-human IgG Fc specificantibody (Jackson Immunoresearch) and plates were carefully washed withPBS containing 0.05% Tween 20 (Biorad). After blocking with PBScontaining 0.1% BSA, in this same buffer, 50 pM of 125I-rhuHGF in 25 mLper well were added. To each well 50 mL of serial dilutions(1:25-1:6000) of cell supernatants, purified CHO-expressed rhuHGF(25,000-0.064 pM) or medium were added in duplicates. Subsequently, 25mL of 50 pM of HGF receptor:IgG fusion protein were added and the plateswere incubated with gentle shaking. After 4 hours, when equilibrium wasreached, plates were washed and wells were individually counted in agamma-counter. The amount of nonspecifically bound radioactivity wasestimated by incubating HGF receptor:IgG with a 500-fold excess ofunlabelled rhuHGF. The dissociation constant (Kd) of each analogue wascalculated at the IC50 from fitted inhibition curves using the huHGFconcentration determined by ELISA.

C. Biological assay

The biological activity of WT huHGF and variants was measured by theirabilities to induce DNA synthesis of rat hepatocytes in primary culture.Hepatocytes were isolated according to published perfusion techniqueswith minor modifications (Garrison and Haynes, J. Biol. Chem. 150,2269-277 (1975)). Briefly, the livers of female Sprague Dawley rats(160-180 g) were perfused through the portal vein with 100 mL of Ca⁺⁺free Hepes buffered saline containing 0.02% Collagenase type IV (Sigma).After 20 minutes the liver was removed, placed in buffer, gently stirredto separate hepatocytes from connective tissue and blood vessels, andfiltered through nylon mesh. Cells were then washed by centrifugation,resuspended at 1×10⁵ cells/mL in Williams Media B (Gibco) containingPenicillin (100 U/ml), Streptomycin (100 mg/mL), L-Glutamine (2 mM),trace elements (0.01%), transferrin (10 mg/mL) and Aprotinin (1 mg/mL).Hepatocytes were incubated in 96-well microtiter plates (Falcon) in thepresence of duplicate serial dilutions of either purified CHO-expressedrhuHGF (1-0.031 mg/mL), 293 supernatants (1:4-1:256) or medium. After 48hours incubation at 37° C., 0.5 mCi 3H-TdR (15 Ci/mmole, Amersham) wasadded to each well and incubated for an additional 16 hours. Cells wereharvested on filter papers, which were washed, dried and counted in aBeckman counter after addition of scintillation liquid. For each hu/HGFvariant, the specific activity (SA) expressed in units/mg was calculatedat half-maximal proliferation (defined as 1 unit/mL) using the HGFconcentration obtained in ELISA.

D. Induction of tyrosine phosphorylations on A549 cells

Human lung carcinoma cells (A549) monolayers were cultured in RPMI 1640medium containing 10% fetal bovine serum and maintained at 37° C. in ahumidified atmosphere with 5% CO₂. Serum-starved cells were incubatedwithout or with 200 ng/mL rhuHGF for 5 minutes at 37° C. and extractedwith lysis buffer containing 50 mM Hepes, 150 mM NaCl, 1.5 mM MgCl₂, 1mM EGTA, 10% Glycerol, 1% Triton X-100 and a cocktail of proteaseinhibitors. The lysates were immunoprecipitated with anti-Met COOHantibodies and blotted with anti-phosphotyrosine antibodies (see Westernblotting above).

EXAMPLE 3 Analysis of Cleavage Site Mutants

The cleavage site of proteases commonly contains a basic residue atposition P1 and two hydrophobic amino acid resides in positions P'1 andP'2, which follow the cleaved peptide bond. The proposed cleavage siteof huHGF (P1 R494, P'1 V495, P'2 V496) fits this consensus. We chose totry to block cleavage of huHGF by replacing the P1 R494 with either D,E, or A. The major form of WT rhuHGF expressed in these cells is cleavedinto two-chain material as judged by the presence of the α-chain with anapparent molecular mass of 69 kDa (FIG. 2). Each of these mutatiomsappeared to block processing of rhuHGF because under reducing conditionsthese variants migrated as a single band at 94 kDa, the predicted sizeof single-chain HGF. These variants totally lacked the ability to inducethe proliferation of hepatocytes in primary culture (FIG. 3A). However,when these variants were analyzed for their ability to compete with WTrhuHGF for binding to the HGF receptor:IgG fusion protein, theirinhibition curves were roughly similar to that of WT rhuHGF (FIG. 3B).The Kd determined from these curves showed that WT rhuHGF binds to thefusion protein with high affinity (50-70pM) whereas all single chainvariants showed approximately a 2- to 10-fold higher Kd (100-500pM)compared to WT rhuHGF. Results from at least three independent assaysare summarized in Table I as residual hepatocyte proliferative activityand receptor binding capacity compared to WT rhuHGF.

Our binding studies showed that WT rhuHGF bound to the soluble receptorfusion protein with a single class of high affinity binding sites (50-70pM), similar to those found on hepatocytes by Higushi and Nakamura(1991). However, binding of HGF on cells may slightly be different sincethe soluble receptor is actually a dimer held together by the disulfidebridge of the hinge in the Fc portion of the IgGA.

Direct comparison of specific activity (SA) versus Kd ratios of allsingle chain variants showed they were inactive at the highestconcentration tested (SA<3%) while receptor binding affinities were onlydecreased by a factor of 2-3.

These results argue strongly that cleavage of HGF into the two-chainform is required for mitogenic activity, i.e. that single-chain HGF is apromitogen and that the uncleaved form of HGF binds to the HGF receptor,albeit with a reduced affinity.

The major form of HGF isolated from placenta (Hernandez et al., (1992)J. Cell Physiol., in press) or expressed in transfected COS cells (Rubinet al., Proc. Natl. Acad. Sci. USA 88, 415-419 (1991)) is insingle-chain form. When tested in mitogenic assays, this single-chainform of HGF is found to be biologically active. Taken together with ourdata, this suggests that this single-chain HGF is activated to thetwo-chain form during the mitogenic assay.

A second observation is that single-chain variants retain substantialcapacity to bind to the HGF receptor, as suggested by our competitionbinding assays. This raises the interesting possibility thatsingle-chain HGF may be bound to cell-surface HGF receptor in vivo in aninactive state and can subsequently be cleaved to the activedouble-chain form by the appropriate protease.

EXAMPLE 4 The Effects of Protease Domain Mutations

To elucidate the functional importance of the protease domain of HGF,several single, double and triple mutations were made in order toreconstitute a potential serine-protease active site. The constructionof these variants is described in Example 1.

We replaced HGF residues Q534 with H, Y673 with S, or V692 with S aseither single, double or triple mutations. The analysis of their effectson mitogenic activity and receptor binding showed that the singlemutation Q534H did not significantly alter either SA (5.2×104 Units/mg)or Kd (60 pM) when compared to wt rhuHGF (respectively 3.3 104 Units/mgand 70 pM) whereas Y673S and V692S exhibited SA reduced approximately 5-and 10-fold, respectively. In fact, these two variants never reached themaximum plateau seen with WT rhuHGF (approximately 50% of wt rhuHGFplateau). Interestingly, these variants showed a Kd similar to WTrhuHGF. All other double and triple variants also retained the abilityto bind the HGF receptor but they clearly showed a reduced SA (Table I).The residual SA of the double variants Q534H,Y673S and Y673S,V692S andof the triple variant Q534H,Y673S,V692S were less than 3% compared to WTrhuHGF. However, the Kd of these variants was not significantlydifferent from WT rhuHGF (Table I). These variants indicate thatmutations within the β-chain of HGF block mitogenic activity but theyare still able to bind to the HGF receptor. Thus, it appears that thesemutants are defective in an activity subsequent to receptor binding.

These results show that although the β-chain is not required forreceptor binding, certain residues (e.g. Y673 and V692) are critical forthe structure and/or activity of HGF. Substitution of the nonpolarresidue V692 with the polar residue S might have caused a structuraltransition if new hydrogen bonds to the active site residue D594, asfound in serine-proteases, have been introduced. Substitution of Y673with the smaller residue S might also introduce some local structuralmodifications. On the other hand, replacement of the polar residue Q534by another polar residue H of similar size would not likely cause adrastic difference in the HGF conformation as this residue should beexposed; indeed the Q534H variant was similar to rhuHGF (Table I).

EXAMPLE 5 The Effect of C-terminal and Kringle Deletions

In order to ascertain whether the α-chain is required for HGF binding oractivity, C-terminal truncations were made as described in Example 1,resulting in variants containing either the α-chain alone, or variantstruncated after the third (N-384) or second (N-303) Kringles.

A number of C-terminal truncations of HGF were made by deleting eitherthe β-chain or the β-chain in addition to a progressive number ofkringles as depicted in FIG. 1. One variant (N-207) corresponding to theN-terminal domain with the first Kringle did not express the protein tolevels detectable either by Western blotting or ELISA using a polyclonalantibody preparation and thus was not investigated further. Expressionof the variants containing the first two Kringles (N-303), threeKringles (N-384) or the complete α-chain of HGF was as low as 250-600ng/mL. A summary of the residual SA and Kd compared to WT rhuHGF ofthese variants is presented in Table I. At the concentration tested noactivity above background levels was observed indicating that thesevariants lost their biological activity. However, binding competitionshowed that variants N-303, N-384 or the α-chain still retainedsubstantial binding capacity (up to 23% compared to WT rhuHGF binding).Thus, the N-terminal 272 residues of HGF (the mature form of variantN-303) are sufficient for high affinity binding to the HGF receptor.Results from deleting each kringle domain are shown in Table I. Deletionof the first Kringle (variant ΔK1) of HGF affected biological activitymost, showing at least a 100-fold reduction (SA<0.2% of wt rhuHGF).Similarly, binding of this variant was also affected as it failed tocompete for binding with wt rhuHGF up to 2 mg/mL. Deletion of all otherKringles (variants ΔK2, ΔK3 or ΔK4) also induces severely reducedmitogenic activity (Table I). However, the Kds of these deletionvariants remained close to that observed with wt rhuHGF.

These data show that Kringles K3 and K4 are not required for receptorbinding. Our data support the previous observations by Miyazawa et al.,1991 supra and Chan et al., 1991 supra, in the sense that variant N-303,which in amino acid sequence is very similar to HGF/NK2, retains theability to compete efficiently for binding to the HGF receptor (Kd˜280pM). Furthermore, the observations that N-303 is sufficient to bind tothe receptor and that the second Kringle is not required for binding theHGF receptor (in the context of the remainder of the molecule) suggestthat the receptor binding domain is contained within the finger andfirst Kringle of huHGF. Unfortunately, we have not been able to detectexpression of this variant using our polyclonal antisera suggesting thatvariant N-207 (deletion after the first kringle) was not expressed in293 cells.

                  TABLE I                                                         ______________________________________                                                       SA var/SA wt Kdwt/Kdvar                                        Variants (var) +/- S.D.     +/-S.D.                                           ______________________________________                                        Single-chain                                                                  R494A          <0.03        0.32 +/- 0.18                                     R494D          <0.03        0.51 +/- 0.21                                     R494E          <0.02        0.31 +/- 0.13                                     Protease                                                                      Q534H          1.19 +/- 0.44                                                                              1.48 +/- 0.85                                     Y673S          0.27 +/- 0.07*                                                                             1.35 +/- 0.72                                     V692S          0.08 +/- 0.04                                                                              1.02 +/- 0.13                                     Q534H, Y673S   <0.03        2.24 +/- 1.11                                     Y673S, V692S   <0.02        1.76 +/- 0.63                                     Q534H, Y673S, V692S                                                                          <0.02        1.91 +/- 1.28                                     C-terminal truncation                                                         N-303          <0.05        0.23 +/- 0.03                                     N-384          <0.05        0.25 +/- 0.02                                     α-chain  <0.04        0.25 +/- 0.03                                     Kringle deletion                                                              ΔK1      <0.002       <0.03                                             ΔK2      <0.05        0.41 +/- 0.18                                     ΔX3      <0.03        0.56 +/- 0.36                                     ΔK4      <0.07        0.86 +/- 0.46                                     ______________________________________                                    

EXAMPLE 6 Induction of Tyrosine-Phosphorylation of the huHGF Receptor

We determined if variants R494E or Y673S,7692S, which bind the HGFreceptor in vitro but are defective for mitogenic activity, couldstimulate tyrosine-phosphorylation of the HGF receptor in A549 cells.Serum starved cells were treated with purified WT rhuHGF or variants andimmunoprecipitates of the HGF receptor were blotted and probed withphosphotyrosine antibodies. Stimulation with wt rhuHGF led to thephosphorylation on tyrosine of the 145 kDa β-subunit of the HGF receptor(FIG. 4). Both variants exhibited a reduced ability to inducephosphorylation of the HGF receptor.

Stimulation of tyrosine phosphorylation on the HGF receptor β-subunit byHGF was previously reported (Bottaro et al., Science 251, 802-804(1991), Naldini et al., 1991 supra). The present data show that variantsR494E and Y673S,V692S can bind the soluble HGF receptor: IgG protein invitro but are not efficient in stimulating tyrosinephosphorylation inA549 cells. One interpretation of this result is that these variants arecapable of binding the HGF receptor on A549 cells, but are defective ina function required to induce efficient phosphorylation, e.g. receptordimerization. It has been shown for other receptor proteins with anintrinsic tyrosine kinase such as the epithelial and platelet-derivedgrowth factor that receptor-receptor interactions or dimerization isrequired for activation of kinase function (see for review Ulrich andSchlessinger, Cell 61 203-212 (1990)). Alternatively, these variants maynot be able to bind the cell-surface associated HGF receptor.

The unique structure of HGF suggests that there may be multiple eventsthat regulate the biological activity of this molecule. An early stageof regulation may be the cleavage step to generate the biologicallyactive two-chain form. Interestingly, cleavage may not simply regulatereceptor binding but rather control a subsequent event required foractivating the HGF receptor. Our data also suggest that the β-chain,while not absolutely required for receptor binding contributes to areceptor activation step. These variants may be useful in dissecting thesignalling events at the HGF receptor.

EXAMPLE 7 Generation and Characterization of HGF/NK1

A. Experimental Procedures

Materials. Heparin-sepharose was purchased from Bio-Rad. Mono Scation-exchange columns and the FPLC equipment were from Pharmacia.SpectraPor/10 dialysis tubing (molecular weight cut off 10,000) was fromSpectrum. All restriction enzymes were obtained from New England Biolabsand used according to manufacturer's instructions. Anti-Flag monoclonalantibody M2 was from IBI, Kodak. Recombinant human HGF was manufacturedin Genentech, Inc., South San Francisco. Purified kringle 4 ofplasminogen was a gift of Frank Castellino.

Bacterial strains. E. coli strain 294 (end A1 thi-1 hsdR F-supE44; ATCC31446) was used for routine transformations and plasmid preparations. E.coli protease-deficient strain 27C7 (tonAD phoADE15 D(argF-laC)169 ptr3degP41 KanR ompTD) was used for expression of HGF/NK1 under control ofthe phoA promoter.

Construction of plasmids and expression of NK1. Plasmid DNA isolation,and polyacrylamide and agarose gel electrophoresis were performed asdescribed (Maniatis et al., Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1982). Bacterial expressionwas performed using the expression plasmid pb0720 (Chang et al., Gene55, 189-196 (1987)) into which was subcloned, 3' of the heat-stableenterotoxin (stII) signal sequence, a modified Flag epitope sequence of10 amino acid residues (S-D-Y-K-D-D-D-D-K-L) (SEQ. ID. No: 26) and themature sequence (Yoshiyama et al., Biochem. Biophys. Res. Commun, 175,660-667 (1991)) of human HGF/NK1 (N-terminal residues 32-210 of humanHGF which contain the complete N- terminal hairpin and kringle 1domains) as outlined in FIG. 8 (the sequences shown in FIG. 8 have beenassigned SEQ. ID. Nos 27, 28 and 29, respectively). The Flag epitope(Hopp et al., Biotechnology, 6, 1205-1210 (1988)) was used to followexpression and secretion of the HGF/NK1. Induction of HGF/NK1 expressionwas performed by phosphate starvation as described (Chang et al.,supra). For subcloning, plasmid pb0720 was digested with NsiI and BamHIand treated with calf intestinal phosphatase. The HGF/NK1 fragment wasisolated by PCR using a specific 5' primer (5'-CCCGGGATGCATCAGACTACAAGGACGACGATGACAAGCTTCAAAGGAAAAGAAGAAAT- 3') (SEQ.ID. NO: 30) encoding a NsiI endonuclease restriction site adjacent tothe Flag epitope sequence and the first six residues of mature HGF. Thereverse 3' primer (5'-CCCGGGAGGCCTCTATTCAACTTCTGAACACTG-3') (SEQ. ID.NO: 31) encoded the last residues of the HGF/NK1 sequence (including the4 residues past the last cysteine of kringle 1 adjacent to a stop codonand a StuI endonuclease restriction site (FIG. 8). Plasmid pRK.huHGFcontaining the complete HGF cDNA sequence was used as a template (Lokkeret al, EMBO J. 11, 2403-2510 (1992)). The isolated Flag-NK1 PCR productwas subsequently digested with NsiI and StuI, and ligated with aStuI-BamHI 1 tO terminator fragment (414 bp; Scholtissek and Grosse,Nucl. Acids Res. 15, 318 (1987) in the above described expressionplasmid pb0720. The generated pf-NK1 plasmid was subsequently sequencedfor verification of the authentic HGF/NK1 sequence using the Sequenasekit (United States Biochemical Corporation). Partial DNA and deducedamino acid sequences of the generated pf-NK1 expression plasmid arediagrammed in FIG. 8.

Purification of NK1. The expression plasmid pf-NK1 outlined in FIG. 8was used to transform E. coli protease-deficient strain 27C7. Thetransformed strain was grown in 10 L of low-phosphate media at 37° C. ina fermenter. Cells were harvested at 36 h after inoculation and storedat -20° C. as a cell paste. Approximately 1 kg of cell paste wasobtained from a 10 L fermenter. A typical purification started from 100g wet weight cell paste and is outlined in FIG. 9. The cell paste wasthawed and suspended in 1 L of ice-cold buffer (10 mM Tris-HCl, pH 7.6,5 mM EDTA, 0.5M NaCl). The suspension was homogenized (tissumizer,Tekmar) and stirred on ice for 1 h. The solution was centrifuged at13,000 rpm for 30 min in a Sorvall GSA rotor. Supernatant was cleared ona 0.2 mM Nalgene filter (with prefilter) and diluted with 10 mM Tris-HCl pH 7.6, 5 mM EDTA, to 0.15M NaCl. The soluble fraction was appliedat 4° C. to an equilibrated Heparin-sepharose affinity column (2.5×10cm) at a flow rate of 20 ml/h. The column was washed with 10 mM Tris-HClpH 7.6, 5mM EDTA, 0.15M NaCl, until the absorbance at 280 nm reachedbaseline values. The bound material was eluted with 10 mM Tris-HCl, pH7.6, 5 mM EDTA, 2M NaCl. The HGF/NK1-containing fractions wereidentified by western blotting using the anti-Flag M2 monoclonalantibody (IBI/Kodak). Positive fractions were pooled, dialyzed against10 mM Tris-HCl pH 7.6, 5 mM EDTA, 0.15M NaCl and applied to a secondheparin column (1×5 cm). Bound proteins were eluted with a lineargradient from 0.15M-2.0M NaCl in 10 mM Tris-HCl pH7.6; 5 mM EDTA. Atthis stage, the HGF/NK1 preparation was judged to be about 80% pure bySDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie andsilver staining. HGF/NK1-containing fractions were pooled, dialyzed in20 mM sodium acetate, pH 6.0, 0.25M NaCl (loading buffer) andchromatographed on a Pharmacia FPLC Mono S cation-exchange column (size5/5). Protein was eluted with a linear gradient from 0.25M-1.5M NaCl ata flow rate of 0.6 ml/min. Fractions between 1 and 1.5 ml werecollected. Positive fractions for HGF/NK1, were pooled and dialyzedagainst 10 mM Tris-HCl pH 7.6, 5 mM EDTA, 0.25M NaCl. Silver staining ofthe final HGF/NK1 sample indicated at least 95% purity. Proteinconcentration was determined by the Bradford method with HGF as astandard and amino acid composition analysis as described below.

Receptor binding, HGF receptor autophosphorylation and biologicalassays. The soluble and A549 receptor binding assays, the induction oftyrosine-phosphorylation on A549 cells, and HGF-stimulated proliferationof hepatocytes in primary culture were performed exactly as described inhe previous examples and in Lokker et al., supra (1992) and Mark et al.,J. Biol. Chem. 267, 26166-26171 (1992). From the binding assays, theapparent dissociation constant of the unlabeled competitor (Kd) wasdetermined using the equation Kd=IC50/(1+(L)/Kd) for competitiveinhibition between two ligands for one type of receptor (Cheng andPrusoff, Biochem. Pharmacol. 22, 3099 (1973)) where IC₅₀ is theconcentration of unlabelled competitor required for 50% displacement of(¹²⁵ I)-labeled HGF binding. (L) is the concentration of theradiolabeled HGF, and the Kd is the apparent dissociation constant for(¹²⁵ I)-HGF.

Isoelectrofocusing (IEF) of NK1. The IEF pattern of HGF/NK1 was examinedon an IEF polyacrylamide gel (pH 3-10) using the Novex gel systemaccording to the vendor's method.

Analysis of proteins by SDS-PAGE. Fractionation of the HGF/NK1 sampleswas performed by SDS-PAGE with glycine-containing 8-16% Tris-glycinegradient gels (Novex) in a Novex minigel apparatus and proteins. Forwestern blotting, proteins were transfered onto nitrocellulose with aPharmacia LKB Biotechnology Inc. Novablot apparatus. The blot wasblocked in 3% dry milk/Tris buffered saline overnight at roomtemperature. The M2 monoclonal antibody (1 mg/ml, IBI/Kodak) was usedfor detection of the Flag-HGF/NK1 fusion protein (2h, room temperature).After three washes in Tris buffered saline, the blot was incubated witha horseradish peroxidase-conjugated antibody to mouse IgG (1:5000,Amersham) for 20 minutes at room temparature and washed four times. Thewestern blot was developed by a chemiluminescent detection system asdescribed by the manufacturer (Amersham).

N-terminal protein sequencing. Automated protein sequencing wasperformed on models 470A and 477A Applied Biosystems sequencers equippedwith on-line PTH analyzers. Electroblotted proteins were sequenced inthe Blot cartridge. Peaks were integrated with Justice Innovationsoftware using Nelson analytical 760 interfaces. Sequence interpretationwas performed on a VAX 8650 J. Chromatogr.: 404, 41-52 (Henzel et al.,(1987)).

Amino acid analysis. Peptides were hydrolyzed for 24 h with 6N constantboiling HCl at 110° C. under vacuum using a Millipore Picotagworkstation. The hydrolysates were dried on a Savant Speed-Vacconcentrator and analyzed on a Beckman model 6300 amino acid analyzerequipped with a ninhydrin detector using a 45 min automated program.

Liquid chromatography/Mass spectrometry(LC-MS). Samples were injectedinto capillary liquid chromatography system (Henzel et al., Anal.Biochem. 187, 228 (1990)) and analyzed directly using a Sciex API IIItriple quadruple mass spectrometer. Multiple charged ions of horsemyoglobilin (MW=16951 kDa) were used for instrument calibration.

Results

Characterization of E. coli-expressed HGF/NK1)--In order to follow theexpression and purification of HGF/NK1 in E. coli, we constructed a genecontaining the coding sequences for immunoreactive `Flag` epitope fusedupstream of residues 32-210 of human HGF as diagrammed in FIG. 8. SinceHGF/NK1 contains 10 cysteine residues, we used a stII leader sequence todirect secretion of the protein into the periplasmic space and purifythe soluble form from the osmotic shock fraction (FIG. 9). Coomassiestaining and western blot analysis using an anti-Flag monoclonalantibody suggested that efficient induction of HGF/NK1 was achieved bygrowing the transformed strain in low-phosphate medium (FIG. 10A and10B). The level of HGF/NK1 expression is estimated between 100-500 mg/L.Using the protocol outlined in FIG. 9, approximately 500 mg of HGF/NK1was purified from the soluble osmotic shock fraction of 100 g of cellpaste. The purified HGF/NK1 from the final FPLC Mono S cation-exchangechromatography step has an apparent molecular weight of 22 kDa asdetermined by SDS- PAGE and is judged to be at least 95% pure (FIG.10C). HGF/NK1 migrates as a monomer as indicated by SDS-PAGE undernonreducing conditions (FIG. 10B). The purified protein was alsoimmunoreactive with a monoclonal antibody to the Flag sequenceconfirming its identity as HGF/NK1 (FIG. 10D).

Biochemical characterization of purified HGF/NK1--The isoelectric point(pI) of HGF/NK1 ranges between 8.2 and 8.6 as judged from the IEF gel(data not shown). The isolated protein has the predicted amino acidcomposition and N-terminal amino acid sequence of correctly processedHGF/NK1 (FIG. 8). The molecular mass was determined to be 21,872 Da byelectrospray ionization mass spectrometry. This number is identical tothe calculated molecular mass of the 32-210 fragment of human HGF linkedto the 10 amino acid Flag epitope.

Receptor binding of HGF/NK1--We assayed the HGF/NK1 preparation forcompetitive binding to a soluble form of the HGF receptor as well as forbinding to the cell-surface associated HGF receptors on A549 cells.Inhibition curves from representative experiments are shown in FIG. 11Aand 11B and indicate that HGF/NK1 is able to compete for the binding ofradiolabeled wild-type HGF to the HGF receptor, albeit with reducedaffinity (8- to 11-fold when compared to wild-type HGF). As expected,purified kringle 4 from human plasminogen did not compete for binding toeither the soluble or cell-associated HGF receptor. Dissociationconstants (Kd) estimated from these curves in at least three independentassays indicate that in solution HGF/NK1 binds with a Kd of 1.10±0.04 nMversus 0.10±0.02 nM for wild-type HGF. Similarly, on A549 cells, HGF/NK1binds with a Kd of 1.6±0.08 nM compared to 0.21±0.04 nM for wild-typeHGF. These data demonstrate that the NK1 region of HGF is sufficient tomediate binding to the HGF receptor.

Ligand-induced of autophosphorylation of the HGF receptor--The HGFreceptor undergoes autophosphorylation of the 145 kDa b-subunit uponbinding of ligand In A549 cells, the maximal response was observed at aconcentration of 1-4 nM (FIG. 12). At these concentrations of HGF/NK1autophosphorylation of the HGF receptor was not detectable. However, athigher concentrations (20 and 100 nM; FIG. 12) some autophosphorylationcould be detected.

Biological properties of HGF/NK1--Whereas HGF stimulates (3H)-thymidineincorporation in primary culture hepatocytes with an half maximal (IB₅₀)effect at 0.64 nM, HGF/NK1 under identical conditions causes noenhancement of DNA synthesis at concentrations as high as 110 nM (FIG.13A). We subsequently tested HGF/NK1 for antagonistic activity usinghepatocytes in primary culture (FIG. 13B). HGF/NK1 completelyantagonizes HGF-induced mitogenic activity with an IB₅₀ of 6 nM,corresponding to a 10-fold molar excess of HGF/NK1 over HGF toneutralize 50% DNA synthesis in hepatocytes. As a control, we showedthat purified kringle 4 of human plasminogen failed to antagonizeHGF-induced mitogenesis. Moreover, the effect of HGF/NK1 was specificsince it failed to neutralize EGF-promoted mitogenesis (data not shown).Thus, HGF/NK1 is a potent and specific antagonist of HGF activity.

EXAMPLE 8 Construction and Expression of HGF-IgG Chimeras

The unique Kpn I site in the coding sequence of wild-type huHGF waslinked to the unique BstE II site of a human IgG-γ1 heavy chain cDNA bya double-stranded synthetic linker (5'-CACAGTCG-3' (SEQ. ID. NO: 21) and5'-GTGACCGACTGTGGTAC-3' (SEQ. ID. NO: 22)). The resulting constructcontained the coding sequences for the entire 728 amino acids of HGFfused by two amino acids (V and T) to amino acids 216-443 of the IgG-γ1heavy chain.

The coding sequences of the HGF variants R494E, and Y673S,V692S(prepared as described in Example 1) were fused in an identical fashionto IgG-γ1.

To construct NK2 HGF-IgG (for brevity also referred to as NK2-IgG), adouble stranded synthetic linker

    5'-ACTGTGCAATTAAAACATGCGAGACG-3'                           (SEQ. ID. NO: 23)

    5'-GTGACCGTCTCGCATGTTTTAATTGCACAGT-3'                      (SEQ. ID. NO: 24)

was used to join the unique Sca I site in IHG to the BstE II site of theIgG-γ1 heavy chain cDNA construct described above. This reconstitutesthe coding sequence of the naturally occurring HGF/NK2 variant describedby Miyazawa et al., supra.

NK1 HGF-IgG (also referred to as NKI-IgG) was constructed by "loop out"deletion mutagenesis using a single-stranded HGF-NK2 template. Themutagenic oligonucleotide used was

    5'-GTCGGTGACCGTCTCTTCAACTTCTGAACA-3'                       (SEQ. ID. NO: 25).

The resulting cDNA contained the coding sequences for amino acids 1-210of HGF joined to those encoding amino acids 216-443 of IgG-γ1 via linkersequences encoding amino acids E, T, V and T.

Expression in 293 cells was performed as hereinabove described.

Control 293 cells and those expressing the HGF-IgG chimeras wereanalyzed by electrophoresis on an 8% SDS-PAGE under reducing (FIG. 5A)and non-reducing (FIG. 5B) conditions. Lane M (Mock) shows that noexpression was detected in control cells. The other lanes represent thefollowing chimeras:

Lane 1: N-303-IgG

Lane 2: HGF-IgG

Lane 3: Y673S,V692S HGF-IgG

Lane 4: R494E HGF-IgG

The results of SDS-PAGE electrophoresis clearly indicate that thechimeras were expressed as dimers.

The proposed structures for some of these HGF-variant-immunoglobulinchimeras are shown in FIG. 5C.

EXAMPLE 9 Binding of HGF-IgG Chimeras to Endogenous HGFr in A549 Cells

The ability of either wild-type recombinant human HGF (wt rhuHGF) or HGFvariant-IgG chimeras to compete for binding of ¹²⁵ I-labeled rhuHGF toA549 cells was studied essentially as described by Naldini, L. et al.,EMBO J. 10, 2867-2878 (1991) with minor modifications. A549 cells,seeded in 24 well plates at a density of 10⁴ cells/well, were grownovernight in DMEM and then shifted to serum free media for 2 hours.Binding was performed with gentle shaking at 4° C. for 3 hours in Hanksmedia containing 20 mM HEPES, 0.2% BSA and 0.02% NAN₃, pH 7.0. Each wellreceived 50 pM ¹²⁵ I-rhuHGF or HGF variant and the indicatedconcentrations of competitor. Extractions and washes were performed asdescribed in Naldini et al., supra.

The results shown in FIGS. 6A and 6B demonstrate that the tested HGF-IgGchimeras bind to endogenous HGFr in A549 cells similar to wild-typehuman HGF (wt rhuHGF).

EXAMPLE 10 Mitogenic Effect on Primary Rat Hepatocyte Cultures

The variant HGF molecules and the HGF variant-IgG chimeras werequantified in the two-site huHGF sandwich ELISA assay described inExample 2A. Conditioned media from 293 cells expressing wt rhuHGF, theindicated HGF variants, and HGF variant-IgG chimeras were tested formitogenic effect on primary rat hepatocyte cultures in the 4H-thymidineuptake assay described in Example 2C. The results are shown in FIGS. 7A,7B and 7C.

As demonstrated in FIG. 7A, conditioned media from cells transfectedwith a plasmid encoding wild-type huHGF stimulated the incorporation of3H in primary rat hepatocyte culture. As shown previously, thesingle-chain HGF variant R494E HGF and the protease domain variantY673S,V692S HGF were defective in mitogenic activity (FIG. 7B).Interestingly however, the mitogenic activity of these variants wascompletely restored when expressed as an IgG fusion protein. Similarly,conditioned media from cells expressing the NK2-IgG variant (FIGS. 7Aand 7C), the NK1-IgG variant (FIG. 7C) but not NK2 or NK1 alone (seeExample 7 for HGF/NK1) also exhibited substantial mitogenic activity.The experiments also show that not all IgG fusion proteins act ashepatic mitogens because the CD4-IgG control failed to induce hepatocyteproliferation. This data are believed to indicate that fusion of the IgGheavy chain region to the HGF variants restores mitogenic activity bycausing these variants to be expressed as dimers.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments without diverting from theoverall concept of the invention. All such modifications are intended tobe within the scope of the present invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 31                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTGGAATCCCATTTACAACCTCGAGTTGTTTCGTTTTGGCACAAGAT47                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAATCCCATTTACGACGTCCAATTGTTTCG30                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCCATTTACAACTGCCAATTGTTTCG26                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGAAGGGAAACAGTGTCGTGCA22                                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGTGGGCCACCAGAATCCCCCT22                                                      (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TCCACGACCAGGAGAAATGACAC23                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCATTCAACTTCTGAGTTTCTAATGTAGTC30                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CATAGTATTGTCAGCTTCAACTTCTGAACA30                                              (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCCATGTGACATATCTTCAGTTGTTTCCAA30                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TGTGGTATCACCTTCATCTTGTCCATGTGA30                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ACCTTGGATGCATTAAGTTGTTTC24                                                    (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TTGTCCATGTGATTAATCACAGT23                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTTCGTGTTGGGATCCCATTTACCTATCGCAATTG35                                         (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AGCTTGCCTCGAGGCA16                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ATGAAGGCCCCCGCTGTGCTTGCACCTGGCATCCTCGTGCTCCTGTTTAC50                          C51                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CACTAGTTAGGATGGGGGACATGTCTGTCAGAGGATACTGCACTTGTCGG50                          CATGAACCGT60                                                                  (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TAGTACTAGCACTATGATGTCT22                                                      (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TTTACTTCTTGACGGTCCAAAG22                                                      (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CAGGGGGAGTTGCAGATTCAGCTGT25                                                   (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AGTTTTGTCGGTGACCTGATCATTCTGATCTGGTTGAACTATTAC45                               (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 bases                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CACAGTCG8                                                                     (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GTGACCGACTGTGGTAC17                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ACTGTGCAATTAAAACATGCGAGACG26                                                  (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GTGACCGTCTCGCATGTTTTAATTGCACAGT31                                             (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GTCGGTGACCGTCTCTTCAACTTCTGAACA30                                              (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      SerAspTyrLysAspAspAspAspLysLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      ATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTTCTAT50                          TGCTACAAATGCCTATGCA69                                                         (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TCAGACTACAAGGACGACGATGACAAGCTTCAAAGGAAAAGA42                                  (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TCAGAAGTTGAATAGAGGTTC21                                                       (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CCCGGGATGCATCAGACTACAAGGACGACGATGACAAGCTTCAAAGGAAA50                          AGAAGAAAT59                                                                   (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CCCGGGAGGCCTCTATTCAACTTCTGAACACTG33                                           __________________________________________________________________________

I claim:
 1. A chimeric molecule comprising a dimer of two monomers eachof said monomers being a fusion protein of a hepatocyte growth factor(HGF) ligand variant to an immunoglobulin constant domain sequence,wherein said dimer is formed by linking the immunoglobulin constantdomain of the two monomers, wherein each monomer alone binds to the HGFreceptor, but wherein at least one monomer alone does not activate thetyrosine kinase activity of the HGF receptor, and wherein the dimerbinds and activates the tyrosine kinase activity of the HGF receptor. 2.The chimeric molecule of claim 1 wherein neither monomer alone activatesthe tyrosine kinase activity of the HGF receptor.
 3. The chimericmolecule of claim 1 wherein said dimer is formed by linking the monomersby a disulfide bond, and wherein each HGF ligand variant is fused at itsC-terminus to the N-terminus of an immunoglobulin constant domaincomprising the hinge region, CH2, and CH3 domains of an IgG-1, IgG-2 orIgG-3 heavy chain.
 4. The chimeric molecule of claim 1 wherein saiddimer is formed by linking the monomers by a disulfide bond, and whereineach HGF ligand variant is fused at its C-terminus to the N-terminus ofan immunoglobulin constant domain comprising the CH 1, hinge region,CH2, and CH3 domains of an IgG-1, IgG-2 or IgG-3 heavy chain.
 5. Achimeric molecules wherein the chimeric molecule is selected from thegroup consisting of NK2-IgG; NK1-IgG; Y673S, V692S HGF-IgG; and R494EHGF-IgG.